CN117642173A

CN117642173A - Methods and kits for inducing immune tolerance to gene delivery targeting agents

Info

Publication number: CN117642173A
Application number: CN202180093128.5A
Authority: CN
Inventors: 吴侠; 仲晨; 肖啸
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2020-12-07
Filing date: 2021-12-06
Publication date: 2024-03-01
Also published as: WO2022121860A1

Abstract

Methods and kits for inducing immune tolerance to an immunogenic target agent in a subject by: administering an immunogenicity inducer vehicle to the subject. Further provided is a method of expressing a target gene in a subject, wherein the subject has been immunocompetent to the immunogenic target agent. Methods of treating diseases are also provided.

Description

Methods and kits for inducing immune tolerance to gene delivery targeting agents

Technical Field

The present disclosure relates generally to the field of gene therapy, and in particular to methods and kits for inducing immune tolerance to a target vehicle for gene therapy in a subject in need thereof. More specifically, the present disclosure relates to methods and kits for inducing immune tolerance to a viral vector carrying a transgene.

Background

Recombinant vectors such as recombinant viral vectors have been widely used as powerful tools for delivering exogenous genes in animals and/or humans and have met with some success in the treatment of human diseases such as duchenne muscular dystrophy (Duchenne muscular dystrophy, DMD), hemophilia B, parkinson's disease, and alpha 1 antitrypsin deficiency.

However, there is an undesirable problem in subjects undergoing gene therapy using these target gene delivery vectors, namely that the host will produce an existing or induced immune response against these potentially immunogenic gene delivery vectors, most notably neutralizing antibodies (NAb) against certain viral packaging proteins (e.g., capsids) and/or transgene products, which severely limits the effectiveness of these therapies, especially repeated gene delivery therapies using the same target gene delivery vector.

In the case of adeno-associated viruses (AAV), while natural AAV is considered nonpathogenic and has the ability to widely infect human tissues and organs, its infection can induce humoral and cellular immunity. Almost half of humans have been shown to possess AAV neutralizing antibodies. In addition, AAV NAb is often induced when one uses AAV gene drugs for the first treatment. These antibodies to AAV capsids and/or transgene products limit the efficacy of recombinant AAV (rAAV) as a drug delivery vehicle. Previous studies showed that NAb generated from the first administration significantly inhibited transgene expression in rabbit lungs from re-administration of rAAV. However, given the limited chemotaxis, seropositive rate, and serotype vector generation, multiple administrations of rAAV gene drugs may be necessary for some genetic diseases that require long-term treatment. Thus, there is a great need to suppress immune responses against AAV to improve rAAV transduction and achieve repeated administration of rAAV during gene therapy. Similar observations were made for other non-AAV vectors.

Disclosure of Invention

In view of the problems of pre-existing or induced host immunity to these gene delivery vectors described above, the present disclosure provides methods and kits for inducing host immune tolerance to an immunogenic gene delivery targeting agent comprising a targeting vector that expresses and encodes a target therapeutic protein or nucleic acid, or more specifically, reducing unwanted host immunity to the immunogenic gene delivery targeting agent.

In a first aspect, a method of inducing immune tolerance to an immunogenic target agent for gene delivery in a subject is provided. The method comprises the following steps: a) Administering an immunogenicity inducer vehicle to the subject.

Herein, the subject administered with the immunogenic target agent may be any animal having an immune response against a foreign immunogen, such as the immunogenic target agent for gene delivery, including a host innate immune response and/or a host adaptive immune response. Optionally, the subject may be an animal capable of producing antibodies, such as a vertebrate. Further optionally, the subject may be a warm-blooded mammal such as primate, dog, cat, cow, horse, sheep, goat, rabbit, rat, mouse, and the like. Further optionally, the subject can be a primate. In certain embodiments, the subject may be a human.

In the methods disclosed herein, the immunogenic inducer vector comprises an inducer nucleic acid vector encoding at least one immunosuppressant, thereby allowing expression of the at least one immunosuppressant in a subject, the at least one immunosuppressant inhibiting an immune response in the subject against the inducer vector. In addition, the immune response is cross-reactive to the target agent.

In certain embodiments of the methods, the immune response comprises a host innate immune response and/or a host adaptive immune response. Optionally, wherein the immune response comprises the generation of at least one antibody capable of binding to the inducer vehicle. Further optionally, the immune response comprises the generation of at least one antibody capable of binding to and neutralizing the inducer vehicle. Further optionally, wherein the at least one antibody comprises one or more antibodies that are cross-reactive with a target agent.

In certain embodiments of the method, wherein the inducer vehicle comprises an inducer viral particle and the target vehicle comprises a target viral particle. Further optionally, wherein each of the inducer viral particle and the target viral particle can comprise a capsid. Further optionally, the immune response of the subject comprises generating at least one antibody capable of binding to the capsid of the inducer viral particle, and further optionally, the at least one antibody is cross-reactive with the capsid of the target viral particle.

In embodiments of the method wherein each of the inducer and target vehicles comprises viral particles, optionally, both the inducer and target viral particles may have the same type of virus, and the type of virus may be selected from the group consisting of: adenoviruses, adeno-associated viruses (AAV), lentiviruses, retroviruses, and oncolytic viruses. In certain embodiments, the type of virus is an adeno-associated virus (AAV).

Herein, the inducer viral particle and the target viral particle may have the same serotype. Alternatively, they may also have different serotypes showing cross-reactivity.

Optionally herein, each of the inducer viral particle and the target viral particle comprises the same capsid protein.

In any of the embodiments of the methods described above, the at least one immunosuppressant can act by downregulating an immunostimulatory pathway and/or by upregulating an immunosuppressive pathway.

In certain embodiments of the method, one or more of the at least one immunosuppressant reduces expression or activity of one or more immunostimulatory modulators in the immunostimulatory pathway, and optionally wherein each of the one or more immunostimulatory modulators is selected from the group consisting of: CD27, CD70, CD28, CD80 (B7-1), CD86 (B7-2), CD40L (CD 154), CD122, CD137L, OX (CD 134), OX40L (CD 252), GITR, ICOS (CD 278) and ICOSLG (CD 275). Herein, the one or more immunostimulatory modulators may be in at least one of the B7/CD28 costimulatory signaling pathway or the CD40/CD40L costimulatory signaling pathway. Furthermore, each of the one or more immunostimulatory modulators may be selected from the group consisting of: CD28, CD80 (B7-1), CD86 (B7-2), CD40 and CD40L (CD 154).

In certain embodiments of the method, one or more of the at least one immunosuppressant increases expression or activity of one or more immunosuppressant modulators in the immunosuppression pathway, and optionally wherein each of the one or more targets is selected from the group consisting of: a2AR, B7-H3 (CD 276), B7-H4 (VTCN 1), BTLA (CD 272), CTLA-4 (CD 152), IDO1, IDO2, TDO, KIR, LAG3, NOX2, PD-1, PD-L2, TIM-3, VISTA, SIGLEC7 (CD 328), TIGIT, PVR (CD 155) and SIGLEC9 (CD 329).

In certain embodiments of the methods, the at least one immunosuppressant modulates the expression or activity of at least one of TLR2, TLR9, myD88, IFN-1, IRF-7, NF- κ B, mTOR, CD3, CD4, CD278, CD19, CD20, CD79, IL-4, IL-2R, IL-5, IL-6R, TNF- α, or LFA-1.

In certain embodiments of the methods, the at least one immunosuppressant down-regulates the B7-CD28 immunostimulatory pathway, and/or down-regulates CD40 and CD40L immunostimulatory pathways. Herein, the at least one immunosuppressant can inhibit binding or signaling of CTLA-4 and CD80 (B7-1) or CD86 (B7-2); and/or may inhibit the binding or signaling of CD40 and CD40L (CD 154).

Furthermore, in certain embodiments, the at least one immunosuppressant may compete with CD28 for binding to B7, or with B7 for binding to CD 28. In other embodiments, the at least one immunosuppressant may compete with CD40 for binding to CD40L, or may compete with CD40L for binding to CD 40.

In the above embodiments of the method, the at least one immunosuppressant may comprise at least one immunosuppressive protein.

Herein, according to certain embodiments, the at least one immunosuppressive protein comprises a CTLA-4 derivative and/or a CD40 derivative. Furthermore, the at least one immunosuppressive protein can optionally include: an extracellular domain of CTLA-4 or a fragment thereof capable of binding to at least one of CD80 (B7-1) or CD86 (B7-2); and/or an extracellular domain of CD40 or a fragment thereof capable of binding to CD40L (CD 154).

In addition, according to certain embodiments, each of the at least one immunosuppressive protein further comprises a half-life extending peptide moiety. Herein, according to certain embodiments, the half-life extending peptide moiety may comprise Fc.

In any of the embodiments of the methods described above, the at least one immunosuppressive agent can include at least one immunosuppressive nucleic acid.

In certain embodiments of the method, step a) further comprises: administering at least one additional inducer vehicle to the subject, wherein each of the at least one additional inducer vehicle comprises an inducer nucleic acid vector encoding at least one additional immunosuppressant. Optionally herein, each of the at least one immunosuppressant and the at least one additional immunosuppressant acts on a different immunomodulator.

In certain embodiments of the method, step a) further comprises: administering to the subject at least one immunosuppressive agent, each immunosuppressive agent capable of inhibiting or enhancing the activity of a target in an immunostimulatory pathway. Herein, each of the at least one immunosuppressive agent can be a compound, a nucleic acid, a polypeptide, or a combination thereof. Further optionally, each of the at least one immunosuppressant and the at least one immunosuppressant agent has a different target.

In any of the embodiments of the claims described above, the targeting agent can include a target nucleic acid vector encoding at least one target gene, each of which can encode a therapeutic protein or therapeutic nucleic acid.

In any of the embodiments of the claims as described above, the subject may have no pre-existing immune response to the inducer vehicle or the target vehicle or both. Optionally herein, the pre-existing immune response may include an immune response to the capsid of the inducer viral particle of the inducer vehicle or to the capsid of the target viral particle of the target vehicle or both.

Optionally, the pre-existing immune response may be characterized by an antibody titer of anti-mediator antibodies of the serum of the subject of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 prior to step a), and wherein the antibody titer is defined as the dilution fold at which the serum of the subject produces a threshold specific binding to the inducer mediator or the target particle. Binding to the inducer vehicle or target vehicle can optionally be measured by an enzyme-linked immunosorbent assay (ELISA). In some embodiments, threshold specific binding is determined based on antibody binding in the presence of a given amount of vehicle, wherein threshold specific binding is considered sufficient to show an antibody binding level with vehicle specific binding.

Further optionally, the pre-existing immune response may be characterized by an antibody titer of neutralizing antibodies of the serum of the subject of at least 2, 4, 6, 8, 18 or 32 prior to step a), and wherein the antibody titer is defined as the dilution factor by which the serum of the subject produces 50% maximum neutralization of the inducer vehicle or the target vehicle. Neutralization of the inducer mediator or target mediator may optionally be measured by a reporter assay.

In any of the embodiments of the methods described above, the immune tolerance may be characterized by an antibody titer of the anti-mediator antibody of the serum of the subject after step a) of no more than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200, and wherein the antibody titer is defined as the dilution factor by which the serum of the subject produces a threshold specific binding to the inducer mediator or the target mediator. Binding to the inducer vehicle or target vehicle can optionally be measured by ELISA assays. In some embodiments, threshold specific binding is determined based on a given amount of vehicle, wherein threshold specific binding is considered sufficient to show a level of antibody binding to the vehicle specific binding.

In any of the embodiments of the methods described above, the immune tolerance may be characterized by an antibody titer of no more than 2, 4, 6, 8, 18 or 32 of neutralizing antibodies in the serum of the subject after step a), and wherein the antibody titer is defined as the dilution of the serum of the subject that produces 50% maximum neutralization of the inducer vehicle or the target vehicle. Neutralization of the inducer mediator or target mediator may optionally be measured by reporter gene detection.

In a second aspect, the present disclosure further provides a method of expressing a target gene in a subject by an immunogenic target agent comprising a target vector encoding the target gene, wherein the subject is tolerogenic to the immunogenic target agent. The method comprises the following steps: a) Administering an immunogenic target agent to the subject. Herein, prior to step a), the existing immune tolerance has been induced by exposing the subject to an immunogenic inducer vehicle comprising an inducer nucleic acid vector encoding at least one immunosuppressant, whereby the at least one immunosuppressant has been expressed in the subject to allow suppression of an immune response directed against the inducer vehicle and cross-reactive with a target viral vehicle.

In a third aspect, the present disclosure further provides a method of expressing a target gene in a subject by an immunogenic target agent comprising a target vector encoding the target gene. The method comprises the following two steps:

a) Administering to the subject an immunogenic inducer vehicle, thereby allowing the at least one immunosuppressant to be expressed in the subject, the immunogenic inducer vehicle comprising an inducer nucleic acid vector encoding at least one immunosuppressant that inhibits an immune response directed against the inducer vehicle and that is cross-reactive with the target vehicle such that immune tolerance is induced in the subject against the target vehicle; and

b) Administering the target agent to the subject having the induced immune tolerance, thereby expressing the target gene in the subject.

In a fourth aspect, the present disclosure further provides a method of treating a condition in a subject with an immunogenic target agent comprising a target vector encoding a target gene capable of treating the condition, wherein the subject is tolerogenic to the target agent. The method comprises the following steps: a) Administering the target agent to the subject, thereby expressing the target gene in the subject to treat the condition. Herein, prior to step a), the existing immune tolerance has been induced by exposing the subject to an immunogenic inducer vehicle comprising an inducer nucleic acid vector encoding at least one immunosuppressant, whereby the at least one immunosuppressant has been expressed in the subject to allow suppression of an immune response directed against the inducer vehicle and cross-reactive with a target vehicle.

In a fifth aspect, the present disclosure further provides a method of treating a condition in a subject with an immunogenic target agent comprising a target vector encoding a target gene capable of treating the condition. The method comprises the following two steps:

a) Administering to the subject an immunogenic inducer vehicle comprising an inducer nucleic acid vector encoding at least one immunosuppressant, thereby allowing expression of the at least one immunosuppressant in the subject, the immunogenic inducer vehicle comprising an inducer nucleic acid vector encoding at least one immunosuppressant that inhibits an immune response directed against and cross-reactive with the inducer vehicle such that immune tolerance is induced in the subject against the target vehicle; and

b) Administering the target agent to the subject having the induced immune tolerance, thereby expressing the target gene in the subject to treat the condition.

In any of the methods provided in the second, third, fourth, and fifth aspects of the disclosure, respectively, as described above, step a) of administering the target agent to the subject comprises repeatedly administering the target agent to the subject.

Further, in any of the methods provided in the second, third, fourth, and fifth aspects of the present disclosure as described above, respectively, various embodiments of the inducer vehicle, the at least one immunosuppressant, the target vehicle, the immune response, and the immune tolerance may refer to various embodiments of the method provided in the first aspect of the present disclosure, a description of which is omitted herein for brevity.

In a sixth aspect, the present disclosure further provides a kit for inducing immune tolerance to an immunogenic target agent to be administered in a subject.

The kit includes a first pharmaceutical composition comprising a therapeutically effective amount of an inducer vehicle, and the inducer vehicle includes at least one inducer nucleic acid vector. Each of the at least one inducer nucleic acid vector encodes and is configured to allow expression of the at least one immunosuppressant in the subject. Each of the at least one immunosuppressant is configured to suppress an immune response in the subject to the inducer vehicle when expressed in the subject, and the immune response is cross-reactive with the target vehicle.

In certain embodiments of the kit, the inducer vehicle comprises an inducer viral particle and the target vehicle comprises a target viral particle. Optionally herein, each of the inducer viral particle and the target viral particle comprises a capsid. The immune response may include the generation of at least one antibody capable of binding to the capsid of the inducer viral particle. The at least one antibody may be cross-reactive with the capsid of the target viral particle.

In certain embodiments of the kit, both the inducer viral particle and the target viral particle have the same type of virus. Herein, the type of virus may be selected from the group consisting of: adenoviruses, adeno-associated viruses (AAV), lentiviruses, retroviruses, and oncolytic viruses. Further, according to some embodiments, the type of virus is an adeno-associated virus (AAV).

Optionally, the inducer viral particle and the target viral particle may be of the same serotype or may be of different serotypes.

In certain embodiments, each of the inducer viral particle and the target viral particle comprises the same capsid protein.

In any of the embodiments of the kits described above, the at least one immunosuppressant acts by downregulating an immunostimulatory pathway and/or by upregulating an immunosuppressive pathway.

In certain embodiments of the kit, one or more immunosuppressants of the at least one immunosuppressant reduces expression or activity of one or more targets in the immunostimulatory pathway, and optionally each of the one or more targets is selected from the group consisting of: CD27, CD70, CD28, CD80 (B7-1), CD86 (B7-2), CD40L (CD 154), CD122, CD137L, OX (CD 134), OX40L (CD 252), GITR, ICOS (CD 278) and ICOSLG (CD 275). In certain embodiments, the one or more targets are in at least one of a B7/CD28 costimulatory signaling pathway or a CD40/CD40L costimulatory signaling pathway. Optionally herein, each of the one or more targets is selected from the group consisting of: CD28, CD80 (B7-1), CD86 (B7-2), CD40 or CD40L (CD 154).

In certain embodiments of the kit, one or more immunosuppressants of the at least one immunosuppressant increases expression or activity of one or more targets in the immunosuppression pathway, and optionally each target of the one or more targets is selected from the group consisting of: a2AR, B7-H3 (CD 276), B7-H4 (VTCN 1), BTLA (CD 272), CTLA-4 (CD 152), IDO1, IDO2, TDO, KIR, LAG3, NOX2, PD-1, PD-L2, TIM-3, VISTA, SIGLEC7 (CD 328), TIGIT, PVR (CD 155) and SIGLEC9 (CD 329).

In certain embodiments of the kit, the at least one immunosuppressant modulates the expression or activity of at least one of TLR2, TLR9, myD88, IFN-1, IRF-7, NF- κ B, mTOR, CD3, CD4, CD278, CD19, CD20, CD79, IL-4, IL-2R, IL-5, IL-6R, TNF- α, or LFA-1.

In any of the embodiments of the kits described above, the at least one immunosuppressant comprises at least one immunosuppressive protein. Optionally herein, the at least one immunosuppressive protein comprises: an extracellular domain of CTLA-4 or a fragment thereof capable of binding to at least one of CD80 (B7-1) or CD86 (B7-2); and/or an extracellular domain of CD40 or a fragment thereof capable of binding to CD40L (CD 154). Further optionally, each immunosuppressive protein of the at least one immunosuppressive protein further comprises a half-life extending peptide moiety. In certain embodiments, the half-life extending peptide moiety comprises Fc.

In any of the embodiments of the kits described above, the at least one immunosuppressive agent comprises at least one immunosuppressive nucleic acid.

In any of the embodiments of the kits described above, each of the inducer vehicle and the target vehicle can comprise a viral particle. In certain embodiments, the viral particles are derived from adeno-associated virus (AAV). In certain embodiments, AAV is produced in a suitable cell line, including but not limited to Hela, HEK293 cell lines, or variants thereof.

In any of the embodiments described above, the kit may further comprise a first instruction for administering the first pharmaceutical composition to the subject.

In any of the embodiments described above, the kit may further comprise a first detection kit for determining whether the immune tolerance is induced in the subject following administration of the first pharmaceutical composition to the subject. Herein, the immune response may optionally include generating at least one anti-mediator antibody and/or at least one neutralizing antibody capable of binding to the inducer mediator and having cross-reactivity with the target mediator, and the first detection kit may include at least one first reagent configured to allow determination of the titer of one or more of the anti-mediator antibodies and/or one or more neutralizing antibodies in the serum of the subject.

In certain embodiments, the kit further comprises a second detection kit comprising at least one second reagent configured to allow detection of expression of each of the at least one immunosuppressant in the subject.

In any of the embodiments described above, the kit may further comprise a second pharmaceutical composition comprising a therapeutically effective amount of the target agent.

Herein, the kit may further comprise a second instruction for administering the second pharmaceutical composition to the subject. The second instructions further comprise a first sub-instruction for administering the second pharmaceutical composition to the subject after administration of the first pharmaceutical composition. The second instructions may further comprise a second sub-instruction for repeated administration of the second pharmaceutical composition.

In certain embodiments of the kit, the targeting agent comprises a targeting vector encoding a target gene and configured to allow expression of the target gene in a subject.

In certain embodiments, the kit further comprises a third detection kit comprising at least one third reagent configured to allow detection of expression of the target gene in the subject.

In a seventh aspect, the present disclosure further provides a kit for expressing a target gene in a subject.

The kit includes a pharmaceutical composition and instructions for administering the pharmaceutical composition. The pharmaceutical composition comprises an immunogenic target agent comprising a target vector encoding the target gene and configured to allow expression of the target gene in the subject. The instructions include a method for inducing immune tolerance to the target agent prior to administration of the pharmaceutical composition to the subject. Herein, the method includes: a) Administering an immunogenicity inducer vehicle to the subject. Herein, an inducer nucleic acid vector comprises an inducer nucleic acid vector encoding at least one immunosuppressant, thereby allowing expression of the at least one immunosuppressant in a subject, the at least one immunosuppressant inhibiting an immune response in the subject against the inducer vector. In addition, the immune response is cross-reactive to the target agent.

The kit may optionally further comprise a first detection kit for determining whether to induce the immune tolerance to the target vehicle in the subject. Herein, the immune response may include generating at least one anti-mediator antibody or at least one neutralizing antibody capable of binding to the inducer mediator and having cross-reactivity with the target mediator, and the first detection kit includes at least one first reagent configured to allow determination of the titer of the anti-mediator antibody and/or one or more neutralizing antibodies in the serum of the subject.

In any of the embodiments of the kit of the seventh aspect, the kit further comprises a second detection kit comprising at least one second reagent configured to allow detection of expression of each of the at least one immunosuppressant in the subject.

In any of the embodiments of the kit of the seventh aspect, the kit may further comprise a third detection kit, wherein the third detection kit comprises at least one third reagent configured to allow detection of expression of the target gene in the subject.

In any of the embodiments of the kit of the seventh aspect, the kit may further comprise instructions, further comprising sub-instructions for repeated administration of the pharmaceutical composition.

In an eighth aspect, the present disclosure further provides a kit for expressing a target gene in a subject by an immunogenic target mediator, the immunogenic target mediator encoding the target gene.

The kit comprises a first pharmaceutical composition for inducing immune tolerance to the target agent in the subject and a second pharmaceutical composition comprising a target agent comprising a target vector encoding a target gene. The first pharmaceutical composition comprises an inducer vehicle comprising at least one inducer nucleic acid vector, each inducer nucleic acid vector encoding and configured to allow expression of at least one immunosuppressant in a subject. Each of the at least one immunosuppressant is configured to suppress an immune response in a subject against an inducer vehicle when expressed in the subject. Here, the immune response is cross-reactive to the target agent.

The kit may further comprise a first detection kit for determining whether the immune tolerance is induced in the subject after administration of the first pharmaceutical composition to the subject. Herein, an immune response includes the production of at least one antibody capable of binding to and cross-reacting with an inducer mediator, and a first detection kit includes at least one first reagent configured to allow determination of the titer of one or more of the at least one antibody in the serum of a subject.

In any of the embodiments of the kit of the eighth aspect, the kit further comprises a second detection kit comprising at least one second reagent configured to allow detection of expression of each of the at least one immunosuppressant in the subject.

In any of the embodiments of the kit of the eighth aspect, the kit may further comprise a third detection kit comprising at least one third reagent configured to allow detection of expression of the target gene in the subject.

In any of the embodiments of the kit of the seventh aspect, the kit may further comprise first instructions for administering the first pharmaceutical composition; and a second instruction for administering the second pharmaceutical composition. Optionally herein, the second instructions may further comprise a first sub-instruction for administering the second pharmaceutical composition to the subject after administration of the first pharmaceutical composition. Further optionally, the second sub-instructions may further comprise a second sub-instruction for repeated administration of the second pharmaceutical composition.

In any of the embodiments of the kits of the sixth, seventh and eighth aspects, the kit may further comprise a third pharmaceutical composition comprising at least one immunosuppressive agent, each immunosuppressive agent being capable of inhibiting or enhancing the activity of a target in the immunostimulatory pathway. Herein, each of the at least one immunosuppressive agent can include a compound, a nucleic acid, a polypeptide, or a combination thereof. In certain embodiments, each of the at least one immunosuppressant and the at least one immunosuppressant agent has a different target.

The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the drawings and the following detailed description.

Drawings

FIGS. 1A-1E illustrate AAV 8-mediated transgene expression in vitro and in vivo, wherein FIG. 1A demonstrates the AAV vector plasmids pAAV-CTLA4-Ig and pAAV-CD40-Ig; FIG. 1B shows Western blotting results of protein CTLA4-Ig or CD40-Ig with molecular weight of 56KD, wherein pAAV-CTLA4-Ig or pAAV-CD40-Ig was transiently transfected into HEK293 cells with PEI, and after 72 hours, the medium and cells were collected for Western blotting with GAPDH as a reference protein for quantification and cells not transfected with plasmid as Negative Controls (NC); FIG. 1C shows Western blotting results for protein CTLA4-Ig or CD40-Ig, wherein AAV8-CTLA4-Ig or AAV8-CD40-Ig viruses infect HUH-7 cells, and after 72 hours, the medium and cells were collected for Western blotting, wherein cells not transfected with the virus were used as NC; FIG. 1D or FIG. 1E shows CTLA4-Ig or CD40-Ig levels in serum as determined by ELISA, wherein two doses of 4X 10 were administered by intravenous injection to C57BL/6J WT male mice of 6-8 weeks of age ¹² vg/kg (high dose, HD) and 4X 10 ¹¹ vg/kg (medium dose, MD) AAV8-CTLA4-Ig or AAV8-CD40-Ig, wherein each value represents mean ± s.d. (n=5); and fig. 1F shows delivery of AAV8 vector to muscle and liver by intravenous injection (i.v.) or intramuscular injection (i.m.), wherein C57BL/6J mice were injected 4 x 10 by hindlimb i.v. or i.m. injection ¹² vg/kg AAV8-GFP and after two weeks, muscle and liver tissue was obtainedGFP fluorescence expression was performed in which muscle frozen sections are shown in sagittal and cross shapes, in which FITC represents GFP, DAPI represents nuclei, untreated mice served as control, and in which the scale is 100. Mu.m.

FIGS. 2A-2D show that CTLA4-Ig and CD40-Ig treatments were effective in inhibiting antibody responses against AAV8 vectors, wherein FIG. 2A shows a study design outline in which, in the first administration, C57BL/6J mice (n=5, each group) were intravenously injected with two doses of AAV8-CTLA4-Ig, AAV8-CD40-Ig, AAV8-GFP or AAV8-CTLA4-Ig+AAV8-CD40-Ig, as described in the tables, 4X 10 ¹² vg/kg (high dose, HD) and 4X 10 ¹¹ vg/kg (medium dose, MD) in which mice using AAV8-GFP were used as control groups, mice not using AAV vector were used as untreated controls, and in the second and third administration experiments at 8 weeks and 15 weeks, respectively, mice in all groups were injected intravenously by 6X 10 ¹¹ vg/kg of AAV8-GFP, then mice were bled weekly after exposure to AAV vector to collect serum for 19 weeks for antibody determination; FIG. 2B shows the results of weekly determinations of AAV8 neutralizing antibody (NAb) titers; FIG. 2C shows the results of an anti-AAV 8 IgG antibody assay, as measured by ELISA, wherein each serum sample was repeatedly assayed; and FIG. 2D shows the correlation between NAb titer and anti-AAV 8 IgG antibody (linear regression: n=5, R=0.8451, p)<0.0001 Where cross means AAV injection time points.

Figures 3A-3D show that AAV8-CTLA4-Ig or AAV8-CD40-Ig immunomodulatory treatment does not impair the humoral immunity of mice, wherein figure 3A shows ELISA results for total mouse IgG in serum, wherein serum samples are collected weekly from mice (n=5, each group) starting with a second administration of AAV vector, and each serum sample is repeatedly tested (n=2); and figures 3B, 3C and 3D show the results of an assay for cytokines including TNF- α, IL-4 and IL-10 in mouse plasma, respectively, determined using Luminex, wherein plasma samples were obtained on the fourth and seventh days of the third administration of AAV vector, respectively, each value representing the mean ± s.d., and each serum sample was repeatedly tested (n=2).

Figures 4A, 4B, 4C, 4D and 4E show transgene expression in liver and muscle by intravenous injection (i.v.) at high or moderate doses via repeated administration of AAV8 vector. Figures 4A and 4B show GFP expression transgenes in which mice were injected as described in figure 2A. At the end of the study, all groups of mice died. Mouse livers, muscles were obtained for GFP fluorescence expression. GFP (shown in green) and nuclei were stained with DAPI (blue). Untreated mice served as untreated control. Scale bar, 100 μm. Figures 4D and 4E show expression transgenes of GLUC, where mice were injected as described in figure 4C, and mouse plasma was collected during the study to measure GLUC expression.

FIGS. 5A-5C show that AAV8-CTLA4-Ig or AAV8-CTLA4-Ig+AAV8-CD40-Ig treatment provides antigen-selective tolerance to AAV8 vectors, and that long-term expression of CTLA4-Ig and CD40-Ig inhibited antibodies to AAV of other serotypes (AAV 843), wherein FIG. 5A shows a summary of a study design in which mice were injected intravenously 4X 10 at the twentieth week of AAV administration ¹² vg/kg AAV843 (AAV 843-GLUC) expressing gaussian luciferase (Gaussia luciferase), and mice were bled weekly to obtain plasma or serum; fig. 5B shows the assay results of NAb titers against AAV8 and AAV843 vectors (×p <0.05，**P<0.01 A) is provided; and figure 5C shows the results of the determination of Relative Light Units (RLU) of GLUC expression in mouse plasma. Each serum sample was tested in duplicate. Each value represents an average ± s.d. (n=5).

FIGS. 6A-6E show the effect of CTLA4-Ig and CD40-Ig immunomodulatory treatments on mouse cellular immunity, wherein CD4 was explored using flow cytometry ⁺ T cells, B220 ⁺ CD19 ⁺ B cell, CD4 ⁺ GL7 ⁺ T cells and CD25 ⁺ FoxP3 ⁺ Modulating the frequency of T cells (tregs), wherein mononuclear cells from Peripheral Blood (PBMC) or spleen of mice that are immunoregulated by treated AAV8-CTLA4-Ig or AAV8-CD 40-Ig: FIG. 6A shows CD4 ⁺ T cells and B220 ⁺ CD19 ⁺ A B cell percentage assay determined at 2 weeks of AAV administration; fig. 6B shows the results of the determination of the percentage of Treg cells, determined at weeks 2 and 7 of AAV administration (×p)<0.01 A) is provided; FIG. 6C shows CD4 ⁺ GL7 ⁺ An assay for the percentage of T cells, the assay being assayed at 17 weeks of administration of AAV; FIGS. 6D-6F show CD4 ⁺ T cells, B220 ⁺ CD19 ⁺ Measurement of the percentage of B cells and tregs, measured at 25 weeks of AAV administration; and FIG. 6G shows B220 in the AAV8-CTLA4-Ig HD group and AAV8-CTLA4-Ig+AAV8-CD40-Ig HD group, respectively ⁺ CD19 ⁺ B cells varied in comparison between 2 and 25 weeks. (n=3-5). * P (P)<0.05，**P<0.01。

Fig. 7A-7B illustrate sequences disclosed in the present disclosure.

Detailed Description

In the following detailed description, reference is made to the accompanying drawings, which are intended to be accompanied by various embodiments provided herein and which are intended to form a part of the description of the embodiments. Like reference numerals in the drawings generally identify like components in the description unless context indicates otherwise.

All descriptions provided herein are intended only to illustrate various embodiments of the invention provided herein and are not intended to be limiting. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the disclosure, and it is to be understood that such equivalent embodiments are to be included herein.

It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. For purposes of illustration, if one embodiment disclosed herein includes components A, B and C, it should be understood that the present disclosure is also intended to include embodiments that contain A, B or C alone, or any combination of A, B or C.

All references cited in this disclosure, including patent applications, issued patents, published articles, or other publications, are incorporated by reference in their entirety for the purpose of providing a method that can be used in conjunction with the description provided herein. For any term presented in one or more publications that is similar or identical to a term explicitly defined in the disclosure, the term explicitly provided in the disclosure controls in all aspects.

Unless defined otherwise specifically, all technical and scientific terms used in this disclosure are generally considered to have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

I.Definition of the definition

Before providing a detailed description of the invention in this disclosure, attention is paid to and defined below.

As used herein, i.e., throughout the disclosure, the articles "a," "an," and "the" are to be construed to mean "one or more" or "at least one" unless otherwise indicated. For example, "a gene" may mean one gene or more than one gene.

As used herein, any numerical range described herein can include the stated range and each number within each subrange unless specifically stated otherwise.

As used herein, the terms "about," "left-right," and the like refer to an amount, level, value, quantity, frequency, percentage, dimension, size, quantity, weight, or length that varies by up to 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference amount, level, value, quantity, frequency, percentage, dimension, size, quantity, weight, or length. In particular embodiments, the term "about" or "approximately" when preceded by a numerical value indicates that the value plus or minus a range of 15%, 10%, 5%, or 1%.

As used herein, the term "polypeptide" is used interchangeably with "peptide," "protein," and the like, and is used to refer to a polymer of amino acid residues or an assembly of polymers of multiple amino acid residues. The term applies to both naturally occurring amino acid polymers and non-naturally occurring amino acid polymers and is to be interpreted as also covering amino acid polymers in which one or more amino acid residues are artificial or synthetic chemical mimics of their corresponding naturally occurring amino acids. The term "protein" generally refers to a large polypeptide. The term "peptide" generally refers to a short polypeptide. Polypeptide sequences are generally described as being amino-terminal (N-terminal) to the left-hand end of the polypeptide sequence and carboxy-terminal (C-terminal) to the right-hand end of the polypeptide sequence.

As used herein, the term "nucleic acid" refers to oligonucleotides or polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and further, should be construed to include equivalents, derivatives, variants, and analogs of RNA or DNA made from nucleotide analogs, single-stranded (sense or antisense) and double-stranded polynucleotides. It is to be understood that the term "nucleic acid" does not refer to or infer a polynucleotide strand of a particular length, and thus nucleotides, polynucleotides and oligonucleotides are also included in the definition.

"percent (%) sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to amino acid (or nucleic acid) residues in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). Conservative substitutions of amino acid residues may or may not be considered the same residue. Alignment for the purpose of determining the percent amino acid (or nucleic acid) sequence identity can be accomplished, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of the national center for biotechnology information (U.S. national Center for Biotechnology Information, NCBI), see also Altschul SF et al, 1990 and Altschul SF et al, 1997), clustalW2 (available on the website of the european bioinformatics institute (European Bioinformatics Institute), see also Higgins DG et al, 1996 and Larkin MA et al, 2007), and ALIGN or Megalign (DNASTAR) software. The default parameters provided by the tool may be used by those skilled in the art or the parameters may be tailored appropriately according to the needs of the alignment, for example by selecting an appropriate algorithm.

As used herein, the terms "include", "comprising", "including", and "containing" mean that the term "includes" is used in a generic sense. "containing (containing)", "having (has)", and the like are synonyms, and are used in an open-ended fashion and do not exclude other elements, features, steps, acts, operations, and the like.

As used herein, the term "or" is used in its inclusive sense (rather than in its exclusive sense) so that, for example, when used in connection with a list of elements, the term "or" means one, some, or all of the elements in the list.

As used herein, the phrase "at least one" means one or more/one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. A phrase referring to "at least one of" a series of items is to be construed as referring to any combination of these items, including individual members. For example, "at least one of A, B or C" is intended to encompass: A. b, C, A and B, A and C, B and C and A, B and C. Unless specifically stated otherwise, a connective language such as the phrase "at least one of X, Y and Z" is otherwise understood by the context generally used to express: the term, etc. may be at least one of X, Y or Z. Thus, such connection language is not generally intended to imply that an embodiment requires the presence of at least one of X, at least one of Y, and at least one of Z, respectively.

As used herein, references to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "additional embodiments," or "another embodiment," or combinations thereof, are understood to mean that a particular feature, structure, or characteristic described in connection with the particular embodiment is included in at least one embodiment of the present disclosure. Thus, the foregoing phrases that exist or appear throughout this disclosure do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Conditional language as used herein, such as "may/may (can, could, might, may)", "e.g. (e.g.)," and the like, is generally intended to convey that certain embodiments include certain features, elements and/or steps, and other embodiments do not include certain features, elements and/or steps, unless explicitly stated otherwise or otherwise understood in the context of use. Thus, such conditional language is not generally intended to imply that features, elements, and/or steps are in any way required for one or more embodiments.

II.Method of inducing immune tolerance to gene delivery targeting agents

In the present disclosure, methods and kits are provided for inducing immune tolerance to a gene delivery targeting agent to be administered to a subject.

While gene delivery may be useful for expressing a target gene in a subject, a vehicle (e.g., a recombinant viral vector) for gene delivery may induce an immune response against the vehicle, thus significantly inhibiting target gene expression in repeated administrations of the gene delivery vehicle. However, repeated administration of gene delivery vehicles may be necessary, for example, for the treatment of some genetic diseases requiring long-term treatment. Accordingly, provided herein are methods and compositions useful for suppressing an immune response to a gene delivery vehicle in order to achieve repeated administration of the gene delivery vehicle and greatly increase target gene expression in a subject.

In certain aspects, the disclosure provides a method of inducing immune tolerance in a subject to an immunogenic target agent for gene delivery. The method comprises the following steps: a) Administering an immunogenicity inducer vehicle to the subject. In other words, an immunogenicity inducer vehicle is administered to induce immune tolerance to a target vehicle that is otherwise immunogenic.

As used herein, the term "tolerability (or immunological tolerance)" is defined as the subject's suppressed immunity to an antigen such that the subject's immune system no longer produces a significant level of immune response to the antigen, i.e., the subject's immune system produces only an acceptably low level of immune response, or even a zero immune response, to the antigen.

The terms "immune response", "immunity" and the like generally refer to the response of a subject in its body to defend against any invading foreign organisms or any substance that the host body deems foreign. In general, the immune response may include a host innate immune response and/or a host adaptive immune response. The host adaptive immune response may further include a dendritic cell, T cell, and B cell response, which may further include antibody production.

As used herein, "T cell" refers to a type of immune cell that contributes to cellular immunity against an antigen. Several T cell types are known, such as CD 4T cells, CD 8T cells, regulatory T cells, etc. "B-cell" refers to a second type of immune cell that can also promote an immune response against a particular antigen, typically by expressing an immunoglobulin that binds to the antigen. Other forms of B cells, i.e., regulatory B cells, can play an inhibitory role in the immune system (Goode I et al, 2014).

In the methods provided herein, both the inducer vehicle and the target vehicle are immunogenic. As used herein, the term "immunogenicity" (immunogenic, immunogenicity) and the like refers to the ability of a particular substance to elicit an immune response in a host in the absence of immune tolerance. In certain embodiments, the host immune response comprises a host innate immune response and/or a host adaptive immune response.

i. Vehicle agent

The term "vehicle" generally refers to a device for delivering a target cargo into a subject. Herein, the term "target cargo" may include any suitable bioactive agent intended to be delivered to a subject for purposes of the present disclosure. Examples of target cargo contained in a vehicle may include nucleic acid vectors encoding transgenes, certain macromolecular entities (e.g., proteins, polypeptides, polynucleotides, etc.), or certain small molecule compounds. After being delivered into a subject, the target cargo may be expressed (if a nucleic acid vector is included) and/or perform its intended function (if a macromolecular entity or a small molecule compound is included).

Any suitable vehicle for delivering the target cargo may be used. In some embodiments, the vehicle is configured for delivery of a nucleic acid vector, and may include, for example, a viral vehicle or a non-viral vehicle. Suitable vehicles in the present disclosure are immunogenic.

In some embodiments, the agent (i.e., the inducer agent and/or the target agent) is a viral agent. Viral vectors are based on viral delivery systems, which may include, for example, recombinant viral particles. In certain embodiments, the vehicle may include a viral particle for delivering the target gene. As used herein, the term "viral particle" or the like means a recombinant viral genome or recombinant viral vector packaged within a viral capsid. The viral genome in the viral particle may be a modified viral genome such that it may lack some of the native viral sequences or have altered some viral sequences and/or may contain some sequences heterologous to the native viral genome. According to various embodiments, the viral genome may be a complete or partial viral genome. Non-limiting examples of viral particles for gene delivery include adenoviruses, adeno-associated viruses (AAV), lentiviruses, retroviruses, herpes Simplex Viruses (HSV), and oncolytic viruses, among others.

In some embodiments, the mediator (i.e., the inducer mediator and/or the target mediator) is a non-viral mediator. The non-viral vehicle is based on a non-viral delivery system, which may include, for example, synthetic nanocarriers. Tremendous advances in gene delivery using nanocarriers have been witnessed in recent years (see, e.g., xu H et al, 2014 and Riley MK et al, 2017). As used herein, the term "synthetic nanocarriers" refers to carriers for target cargo (i.e., polynucleotide agents such as nucleic acid carriers, polypeptide agents, antibodies, small molecule agents, etc.) to be delivered to a subject that are not present in nature and have at least one dimension that is less than or equal to 5 microns in size. In some embodiments, the nanocarriers or nanoparticles have a diameter of less than 1000nm.

Synthetic nanocarriers may include, but are not limited to, lipid-based nanoparticles (i.e., nanoparticles whose majority of the material comprising their structure is lipid), polymer nanoparticles, microparticles, microcapsules, metal nanoparticles, synthetic liposomes, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e., particles consisting essentially of viral structural proteins but not having infectivity or having low infectivity), peptide-or protein-based particles (also referred to herein as protein particles, i.e., particles whose majority of the material comprising their structure is peptide or protein) (e.g., albumin nanoparticles), and/or nanoparticles developed using a combination of nanomaterials such as lipid-polymer nanoparticles. Examples of such nanocarriers or nanoparticles may include liposomes (see, e.g., WO 2012/149755; WO 2012/149252) or dextran particles (see, e.g., U.S. patent publication nos. 2005/0281781 and 2010/0040656 and WO 2006/007572 and WO 2007/050643).

Synthetic nanocarriers can take a variety of different shapes including, but not limited to, spherical, cubic, pyramidal, elliptical, cylindrical, annular, and the like. The synthetic nanocarriers may comprise one or more surfaces. The synthetic nanocarriers may be solid or hollow and may include one or more layers. In some embodiments, each layer has a unique composition and unique properties relative to the other layers. By way of example only, the synthetic nanocarriers may have a core/shell structure in which the core is one layer (e.g., a polymer core) and the shell is a second layer (e.g., a lipid bilayer or monolayer). The synthetic nanocarriers may comprise a plurality of different layers.

There are a variety of compositions that can be used to synthesize nanocarriers, which are well known to those skilled in the art and reference can be made to the following documents: U.S. Pat. No. 5,543,158, U.S. patent application No. US20060002852, U.S. 20090028910, U.S. Pat. No. 20020086049, U.S. Pat. No. 20080145441, U.S. Pat. No. 20090226525, U.S. Pat. No. 20060222652 and U.S. Pat. No. 20060251677, international patent application Nos. WO 2010047839A1, WO2009/051837 and WO 2009106999A2 and publications Paolicelli P et al, 2010 and Look M et al 2013, etc. In general, synthetic nanocarriers may include one or more lipids (e.g., liposomes, lipid bilayers, lipid monolayers, micelles, etc.), certain particles (e.g., metal particles, quantum dots, ceramic particles, etc.), one or more amphiphilic entities, polymers, and/or one or more carbohydrates.

Cross-reactivity

In certain embodiments, the immune response to the inducer vehicle is cross-reactive with the target vehicle. The term "cross-reactivity" in relation to an immune response generally refers to the situation where an immune response initially directed against a first entity is also reactive towards a second entity. Herein, the first entity and the second entity may be an inducer mediator and a target mediator. Thus, the subject's immune response (e.g., antibodies raised in the host body) against the inducer vehicle may be "cross-reactive" in that it can also recognize and bind to the target vehicle. In certain embodiments, the inducer vehicle and the target vehicle share at least one common or similar immunogenic antigen or epitope such that upon administration of the inducer vehicle, antibodies are produced in the subject against one or more of the at least one common or similar immunogenic antigen or epitope found in the target vehicle. These antibodies in the subject should also be able to recognize and target shared immunogenic antigens or epitopes upon administration of the targeting agent, thereby generating cross-reactivity. It is noted, however, that the immunogenic antigen from the inducer vehicle and the immunogenic antigen from the target vehicle do not necessarily have to be identical for cross-reactivity of the host immune response. According to certain embodiments, such cross-reactivity may be caused by common or similar epitopes from different immunogenic antigens of the inducer vehicle and the target vehicle.

In one particular embodiment, described in further detail in example 1 below, both the inducer mediator and the target mediator are adeno-associated virus (AAV) based and they share at least the capsid protein of AAV or an epitope in the capsid protein as a common immunogenic antigen or epitope. Thus, a host immune response developed in a subject against a capsid protein or epitope of an inducer vehicle following administration of the inducer vehicle is also cross-reactive with a subsequently administered target vehicle.

Without wishing to be bound by any theory, it is believed that by administering an inducer vehicle that delivers at least one immunosuppressant to a subject, the subject's host immune response to the inducer vehicle is inhibited, resulting in immune tolerance not only to the inducer vehicle but also to the target vehicle. Thus, the effectiveness and efficiency of target agent delivery in the same subject can be improved.

In certain embodiments, each of the inducer vehicle and the target vehicle comprises a viral particle. In some embodiments, the viral particle is in the form of an infectious virion. The viral particles may be based on any virus therapeutically suitable for gene delivery, and non-limiting examples include adenoviruses, adeno-associated viruses (AAV), lentiviruses, retroviruses, oncolytic viruses, and the like. Viral particles include nucleic acid vectors that include sequences encoding one or more target genes (i.e., transgenes or immunosuppressives).

In some embodiments, the inducer viral particle and the target viral particle can have the same type of virus, e.g., the same type of virus can be an adenovirus, adeno-associated virus (AAV), lentivirus, retrovirus, or oncolytic virus. In some embodiments, both the inducer viral particle and the target viral particle are adeno-associated viruses (AAV).

In some embodiments, the inducer viral particle and the target viral particle may have the same serotype. As used herein, the term "serotype" with respect to a viral particle refers to reactivity with a defined antiserum. Serotypes can be used to subgroup virus particles to subspecies levels and generally indicate that virus particles with the same serotype can share the same surface antigen or share surface antigen epitopes.

In some embodiments, the inducer viral particle and the target viral particle may have different serotypes, but still exhibit cross-reactivity.

In certain embodiments, both the inducer viral particle and the target viral particle comprise capsids. As used herein, the term "capsid" refers to the protein envelope of a virus that encapsulates viral nucleic acid. Typically, the capsid comprises several oligomeric structural subunits (i.e., precursors). Herein, the capsid is immunogenic in the subject.

In some embodiments, the inducer viral particle and the target viral particle both comprise the same capsid protein.

For more details regarding the various viruses listed above, reference may be further made to the following section.

In certain embodiments, the immune response to the inducer vehicle comprises the production of at least one antibody that is capable of binding to the inducer vehicle.

In certain embodiments, the immune response comprises the generation of at least one antibody capable of binding to and neutralizing the inducer vehicle. As used herein, the term "neutralising (neutralizing, neutralize, neutralization)" and the like refer to the effect of immunoglobulins (including antibodies such as those produced in a host immune response) that reduce the efficiency of delivery of an inducer vehicle. For example, in embodiments in which the inducer vehicle comprises a viral particle, neutralization may be achieved such that the at least one antibody produced in the subject is directed to the surface of the virion (e.g., capsid protein), which may result in aggregation of the virion, or may be achieved by inhibiting fusion of the virus and cell membrane after attachment of the viral particle to the target cell, inhibiting endocytosis, inhibiting progeny virus from the infected cell, and the like. Regardless of the mechanism, the at least one antibody that exerts a "neutralizing" effect in the subject's host immune response results in a significant decrease or even complete attenuation in the delivery efficiency of the inducer vehicle.

In certain embodiments, the immune response includes the generation of antibodies that bind to and/or neutralize the inducer and target vehicles.

In certain embodiments, the immune response to and cross-reactivity with the inducer vehicle comprises generating at least one antibody that is capable of binding to the capsid of the inducer viral particle and the capsid of the target viral particle.

immunosuppressant agent

In the methods provided herein, an immune response to an inducer vehicle is inhibited. Inhibition of the host adaptive immune response may involve inhibition of T cell and/or B cell responses, and in some embodiments, may involve inhibition of antibody production. This suppressed immune response is directed against the inducer vehicle, but may be cross-reactive with the target vehicle.

The immune response to the inducer vehicle can be inhibited by the immunosuppressant. As used herein, the term "immunosuppressant" refers to a biologically active molecule that has an immunosuppressive effect upon delivery to a subject, resulting in inhibition of a host immune response including a host innate immune response and/or a host adaptive immune response. In certain embodiments, the immunosuppressant may comprise a protein, peptide, nucleic acid, or chemical compound.

In certain embodiments, the immunosuppressant is delivered by an inducer vehicle. As used herein, the phrase "delivered by..a" in reference to an immunosuppressant broadly encompasses embodiments in which the immunosuppressant or its encoding nucleic acid is associated with an inducer vehicle and delivered to a subject in such associated form. For example, the immunosuppressant may be encapsulated in or conjugated to an inducer vehicle and delivered in such encapsulated or conjugated form, after which the immunosuppressant may optionally be released. For another example, a nucleic acid encoding an immunosuppressant can be delivered by an inducer vehicle, and after delivery can be allowed to express or produce the immunosuppressant in a subject.

In certain embodiments, the inducer vehicle comprises an inducer nucleic acid vector encoding at least one immunosuppressant that, upon expression in a subject, inhibits an immune response in the subject against the inducer vehicle. In some embodiments, the inducer vehicle can include one or more inducer nucleic acid vectors. In some embodiments, the inducer nucleic acid vector can encode one or more than one immunosuppressant, depending on the actual need and the ability of the inducer nucleic acid vector.

In some embodiments, the inducer vehicle can include an inducer viral particle that includes the inducer nucleic acid vector.

In some other embodiments, the inducer vehicle can include a non-viral nanocarrier, such as a synthetic nanocarrier capable of delivering an immunosuppressant or an inducer nucleic acid vector.

As used herein, an "immunostimulatory pathway" generally refers to a signaling pathway that has a stimulatory effect on the immune system of a host, which may forward regulate cytokine secretion, NK cell activation, T cell proliferation, antibody production, and the like. Immunostimulatory modulators found in the immunostimulatory pathway may include CD27, CD70, CD28, CD80 (B7-1), CD86 (B7-2), CD40L (CD 154), CD122, CD137L, OX (CD 134), OX40L (CD 252), GITR, ICOS (CD 278), ICOSLG (CD 275), and the like. Any of these immunostimulatory modulators may be inhibited by an immunosuppressive agent provided herein, such that once delivered to and optionally expressed in a subject by an inducer vehicle, the immunosuppressive agent may reduce expression of the immunostimulatory modulator or reduce activity of the immunostimulatory modulator, resulting in an inhibition of an immune response against the inducer vehicle, resulting in immune tolerance to the target vehicle.

In certain embodiments, the at least one immunosuppressant can inhibit expression and/or function of an immunostimulatory modulator that is involved in the B7/CD28 and/or CD40/CD40L costimulatory signaling pathways. According to certain embodiments, the immunostimulatory modulator may be selected from the group consisting of: CD28, CD80 (B7-1), CD86 (B7-2), CD40 and CD40L (CD 154).

As used herein, an "immunosuppressive pathway" generally refers to a signaling pathway in a host that has an inhibitory effect on the host's immune system, which may reverse modular cytokine secretion, NK cell activation, T cell proliferation, antibody production, and the like. Immunosuppression modulators in the immunosuppression stimulation pathway may include A2AR, B7-H3 (CD 276), B7-H4 (VTCN 1), BTLA (CD 272), CTLA-4 (CD 152), IDO1, IDO2, TDO, KIR, LAG3, NOX2, PD-1, PD-L2, TIM-3, VISTA, SIGLEC7 (CD 328), TIGIT, PVR (CD 155), or SIGLEC9 (CD 329), etc. Any of these immunosuppressive modulators can be stimulated by an immunosuppressive agent provided herein, such that the immunosuppressive agent, once delivered to and optionally expressed in a subject by an inducer vehicle, can increase expression of the immunosuppressive modulator or enhance activity of the immunosuppressive modulator, resulting in suppression of an immune response against the inducer vehicle, resulting in immune tolerance to the target vehicle.

According to certain embodiments, the at least one immunosuppressant may increase expression and/or activity of an immunosuppression modulator selected from the group consisting of: TLR2, TLR9, myD88, IFN-1, IRF-7, NF-. Kappa. B, mTOR, CD3, CD4, CD278, CD19, CD20, CD79, IL-4, IL-2R, IL-5, IL-6R, TNF-. Alpha.and LFA-1.

Details of these above-mentioned modulators in the immunostimulatory and immunosuppressive pathways are known in the art, see, for example, WO 97/28259; WO 98/16247; WO 99/11275; krieg AM et al, 1995; yamamoto S et al, 1992; ballas ZK et al, 1996; kliman DM et al, 1997; sato Y et al, 1996; pisetsky DS,1996; shimada S et al, 1986; cowdery JS et al, 1996; roman H et al, 1997; lipford GB et al, 1997; WO 98/55495 and WO 00/61151, etc.

In certain embodiments, the at least one immunosuppressant down-regulates the B7-CD28 immunostimulatory pathway. In certain embodiments, the at least one immunosuppressant inhibits the expression and/or function of CD28, CD80 (B7-1), CD86 (B7-2). In certain embodiments, the at least one immunosuppressant disrupts or blocks B7-CD28 interactions (e.g., binding). In certain embodiments, the at least one immunosuppressant competes with CD28 for binding to B7, or competes with B7 for binding to CD 28. For example, the at least one immunosuppressant may comprise a CD80 binding peptide or a CD80 binding fragment of an anti-CD 80 antibody, a CD28 binding peptide or a CD28 binding fragment of an anti-CD 28 antibody, or a CD86 binding peptide or a CD86 binding fragment of an anti-CD 86 antibody.

In certain embodiments, the at least one immunosuppressant comprises a CTLA-4 derivative. CTLA-4 or CTLA4, known as cytotoxic T lymphocyte-associated antigen 4, is a natural molecule that down-regulates the B7-CD28 immunostimulatory pathway. Exemplary sequences for full length human CTLA-4 are shown in SEQ ID NO. 1 (with signal peptide) and SEQ ID NO. 15 (without signal peptide), and full length mouse CTLA-4 is shown in SEQ ID NO. 12 (with signal peptide). CTLA-4 is an inhibitory receptor expressed on the cell surface of T cells. CTLA-4 is a homolog of CD28, but it has a 100-fold affinity for B7 over CD28, so CTLA-4 expression can cause competitive binding to CD80 and CD86, block B7-CD28 interactions and terminate T cell activation (Li W et al 2009, dall' era M et al 2004 and Slavik JM et al 1999). As used herein, the term "CTLA-4 derivative" is intended to encompass all analogs, mutants and fragments of CTLA-4 as well as such fusion polypeptides that can bind to B7 and compete with CD28 for binding to B7.

In certain embodiments, the at least one immunosuppressant comprises an extracellular domain of CTLA-4 or a B7 binding fragment thereof. B7 as used herein includes B7-1 and B7-2.

As used herein, the phrase "extracellular domain (ECD) of CTLA-4" broadly encompasses wild-type ECD CLTA-4 and all variants, mutants, derivatives or multimers (e.g., dimers, tetramers, hexamers) thereof, so long as they are functionally equivalent to wild-type ECD CTLA-4. The term "functionally equivalent" means that variants, mutants and derivatives of ECD CTLA-4 still retain at least part of the function of the wild-type counterpart, e.g. retain at least part of the function of binding to a natural ligand such as B7. Functional equivalents may have weaker (but not negative), substantially similar, or even higher functions relative to wild-type counterparts. The ECDs of CTLA-4 provided herein can be mammals such as humans or mice. Exemplary amino acid sequences for wild-type ECDs of human CLTA-4 include SEQ ID NO. 11 (ECD of full length human CLTA-4, NO signal peptide, uniProtKB-P16410, amino acids 36-161) and SEQ ID NO. 3 (ECD of human CLTA-4, uniProtKB-P16410, amino acids 38-161). An exemplary amino acid sequence for a wild-type ECD of mouse CLTA-4 is shown in SEQ ID NO. 13.

Different mutants have been shown to be useful in retaining B7 binding activity. In certain embodiments, the ECD of human CLTA-4 in the immunosuppressant comprises one or more mutations at a position selected from the group consisting of: a29, L104, K28, T30, R33, T51, Y52, M53, M54, G55, N56, E57, L58, L61, K93, L96, Y99 and or any combination thereof, wherein the numbering is relative to SEQ ID No. 3.

In certain embodiments, the ECD of human CLTA-4 in the immunosuppressant comprises one or more mutations at a position selected from the group consisting of: L104E, A29Y, G A, K H/T, A29H/T/Y/W/K, T G/E, R33D, T Y/N, Y52F, M F/Y/W, M54R/W/Y, G55P, N56T, E57P, L G/A/N/Q/E/P/Y/H/K, L61H/Y/A/F/W, D62H, K M/V/Q/I, L96K/R, L104H/Y/I/M and any substitution at Y99 or any combination thereof, wherein the numbering is relative to SEQ ID NO 3. These exemplary mutations in the ECD of CTLA-4 are reported to exhibit B7 binding activity, see Z.xu et al, J immunol.) "2012, 11/1/189 (9) 4470-4477; U.S. patent No. US 8,148,332; US 8,642,557; US 5,773,253, 6,090,914; no. 5,844,095; PCT publication No. WO 01/92377A 2.

In certain embodiments, the at least one immunosuppressant can comprise a B7 binding fragment of the ECD of CTLA-4, so long as it still allows high affinity binding to B7 (i.e., CD80 and/or CD 86).

In certain embodiments, the B7 binding fragment of CTLA-4 can be fused to a fragment of another protein, together rendering such hybrid fusion proteins exhibit high B7 affinity. For example, an ECD fragment of CTLA-4 may be fused to another fragment of the ECD of CD28 to allow the resulting hybrid fusion polypeptide moiety to have a high affinity for B7 (see U.S. Pat. No. 6,090,914). An exemplary ECD of the CD28 wild-type amino acid sequence is shown in SEQ ID NO:2 (UniProtKB, P10747, amino acids 19-220).

In certain embodiments, the extracellular domain of CTLA-4 comprises the amino acid sequence shown as SEQ ID NO. 3 or SEQ ID NO. 11, or a sequence having at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO. 3 or 11 but retaining binding activity or affinity for B7-1 or B7-2.

In certain embodiments, the at least one immunosuppressant down-regulates the CD40/CD40L immunostimulatory pathway. In certain embodiments, the at least one immunosuppressant inhibits expression and/or function of CD40 or CD 40L. CD40 and CD40L are located on the surface of Antigen Presenting Cells (APC) and T cells, respectively. When the CD40 and CD40L interact, B7 is strongly expressed in the T cells, which are then activated (Dall' Era M et al, 2004). Exemplary sequences for full length human CD40 are shown in SEQ ID NO. 16 (with signal peptide) and SEQ ID NO. 17 (without signal peptide).

In certain embodiments, the at least one immunosuppressant disrupts or blocks CD40 and CD40L interactions. In certain embodiments, the at least one immunosuppressant competes with CD40L for binding to CD40, or competes with CD40 for binding to CD 40L. For example, the at least one immunosuppressant may comprise a CD40 binding peptide or a CD40 binding fragment of an anti-CD 40 antibody, a CD40L binding peptide or a CD40L binding fragment of an anti-CD 40L antibody.

In certain embodiments, the at least one immunosuppressant comprises a CD40L binding ligand or a CD40L binding fragment of an anti-CD 40L antibody. Exemplary anti-CD 40L antibodies include, but are not limited to, MR1 and BG9588 (see, e.g., noelle RJ et al, 1992 and Boumpas DT et al, 2003). These anti-CD 40L antibodies are believed to be useful for stabilizing transgene expression and enhancing effective re-administration by adenovirus vectors (Jiang Z et al, 2004, chirmule N et al, 2000, yang Y et al, 1996a and Yang Y et al, 1996 b).

In certain embodiments, the at least one immunosuppressant comprises a CD40 derivative. As used herein, the term "CD40 derivative" is intended to encompass all analogs, mutants and fragments of CD40 as well as such fusion polypeptides that can bind to CD40L and compete with CD40 for binding to CD 40L. In certain embodiments, the at least one immunosuppressant comprises an extracellular domain of CD40, a CD 40L-binding fragment of CD40, or a mutant thereof that retains CD40L binding function or affinity.

The extracellular domain portion of CD40 may be wild-type or may have other options reported below: U.S. patent No. 6,376,459 and U.S. patent publication No. 20140120091 A1; elgueta R et al, 2009; clark EA et al, 1986; and Noelle RJ et al, 1992. In certain embodiments, the extracellular domain of CD40 comprises a sequence as set forth in SEQ ID NO:4, or a sequence having at least 70% (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO:4 but retaining binding activity or affinity for CD 40L. An exemplary sequence of the ECD of mouse CD40 is shown in SEQ ID NO. 14.

In certain embodiments, the at least one immunosuppressant down-regulates both the B7-CD28 immunostimulatory pathway and the CD40/CD40L immunostimulatory pathway. In certain embodiments, the at least one immunosuppressant comprises a first immunosuppressant which down-regulates the B7-CD28 immunostimulatory pathway and a second immunosuppressant which down-regulates the CD40/CD40L immunostimulatory pathway. Down-regulation of the B7-CD28 immunostimulatory pathway may involve inhibition of expression and/or function of CD28, CD80 (B7-1), or CD86 (B7-2) or disruption or blocking of B7-CD28 interactions (e.g., binding). Down-regulation of the CD40-CD40L immunostimulatory pathway may involve inhibiting expression and/or function of CD40 or CD40L or disrupting or blocking CD40-CD40L interactions.

The generation of neutralizing antibodies (nabs) as induced by gene delivery vectors (e.g., AAV) is believed to be associated with T cell and B cell activation (Wang D et al, 2019). B7/CD28 and CD40/CD40L signaling pathways co-stimulate T and B lymphocyte activation and play an important role in graft rejection and autoimmune diseases (Arima T et al, 1996). These signaling pathways determine the magnitude of T cell responses to antigens as well as downstream responses to antigens (see, e.g., mueller DL et al, 1989; bessis N et al, 2004).

In certain embodiments, the at least one immunosuppressive agent comprises at least one immunosuppressive protein, which may comprise one of the following, or a combination of two immunosuppressive proteins comprising:

(1) An extracellular domain of CTLA-4 or a fragment thereof capable of binding to at least one of CD80 (B7-1) or CD86 (B7-2); and

(2) The extracellular domain of CD40 or a fragment or antibody fragment capable of binding to CD40L (CD 154).

In certain embodiments, one or more of the immunosuppressive proteins further comprises a half-life extending moiety, e.g., a half-life extending peptide moiety.

In certain embodiments, the at least one immunosuppressant comprises an extracellular domain of CTLA-4, optionally further linked to a half-life extending peptide moiety. In certain embodiments, the at least one immunosuppressant comprises an extracellular domain of CD40, optionally further linked to a half-life extending peptide moiety.

As used herein, the term "half-life" refers to the pharmacokinetic properties of a particular molecule of interest that are intended to measure the survival time of the molecule after its administration. Half-life may be expressed as the time required to eliminate fifty percent (50%) of a known amount of a molecule from the subject's body or a particular compartment thereof, e.g., as measured in serum, i.e., circulatory half-life, or in other tissues. As used herein, a "half-life extending peptide moiety" refers to a peptide fragment or peptide moiety operably linked or fused to a target polypeptide moiety of interest, i.e., an immunosuppressant, which can increase or extend the half-life of an entire fusion or chimeric polypeptide expressed in a particular compartment (especially the circulation) of a subject's body. In other words, upon administration of an inducer vehicle comprising an inducer nucleic acid vector encoding such an engineered immunosuppressant to a subject, the half-life extending peptide moiety can increase the half-life of the fusion polypeptide in the subject, particularly the serum or plasma half-life in vivo.

Exemplary half-life extending peptide moieties include, but are not limited to, fc (i.e., fragment crystallizable regions, such as the Fc of IgG), albumin family members (i.e., albumin (e.g., human serum albumin or HSA), afaxin, alpha fetoprotein, vitamin D binding protein, etc.) or functional (i.e., capable of extending half-life) fragments thereof, serum albumin binding polypeptides (e.g., serum albumin binding antibody fragments, domain antibodies, etc.), follicle Stimulating Hormone (FSH) and functional fragments thereof, fibronectin or functional fragments thereof, fibroblast Activating Protein (FAP) or functional fragments thereof, or clotting factors (e.g., von willebrand factor (von Willebrand factor), factor V, and prothrombin factors, including factor VII, factor IX, factor X, protein IX, protein S, protein Z, and prothrombin), or functional fragments thereof, etc. Prior art disclosing such half-life extending peptide moieties includes WO 2001/079271; WO 2005/024944; WO 2004/101740; WO 2013/106787; and WO 2008/077616; U.S. patent No. 7,625,564; 8,546,543; and 9738707, etc.; and Beattie et al, 1982; lichenstein et al, 1994 and Cooke NE et al, 1985, et al.

According to certain embodiments, the half-life extending peptide moiety comprises an Fc moiety. Herein, the Fc portion may be derived from any of IgG1, igG2, igG3, igG4, and may be wild-type or contain variations or combinations thereof, including T250Q/M428L, V308P, M428L, M Y/S254T/T256E, M428L/N434S, N434A and N434H (numbered according to the EU index of Kabat et al, 1991, and EU numbering scheme used below). These Fc variants are reported to increase the affinity of IgG to neonatal Fc receptors (FcRn) that plays a key role in regulating IgG homeostasis (see, e.g., datta-Mannan a et al, 2007; datta-Mannan a et al, 2012; hiton PR et al, 2006;Dall'Acqua WF et al, 2006; zalevsky J et al, 2010; deng R et al, 2010; maeda a et al, 2017, etc.). In addition, the Fc portion can be an artificial heterodimeric Fc variant, which can include the following designs (KiH, kiHS-S, HA-TF, ZW1, DD-KK, EW-RVT, EW-RVTS-S, SEED, A107). For more details, see review and research article Ha JH et al, 2016; wei H et al, 2017; atwell S, et al, 1997; von Kreudenstein TS et al, 2013; gunasekaran K et al 2010; choi HJ et al, 2013; and Klein C et al 2012.

In certain embodiments, the at least one immunosuppressant comprises a CTLA-4 extracellular domain fused to an Fc, optionally via a linker (e.g., a peptide linker). Alternatively or additionally, in certain embodiments, the at least one immunosuppressant comprises a CD40 extracellular domain fused to Fc, optionally via a linker (e.g., a peptide linker).

These above fusion proteins, i.e., CTLA4-Ig or CTLA4-Fc and CD40-Ig or CD40-Fc, are known in the art. See, e.g., najafian N et al, 2000 and Kirk AD et al, 1997. Two commercially available CTLA4-Ig fusion proteins as described above include abatacept and belatacept, which are approved for the treatment of autoimmune diseases such as rheumatoid arthritis. According to certain embodiments, the Fc portion of the fusion polypeptides of immunosuppressants (i.e., CTLA4-Ig and CD 40-Ig) can have the sequence shown in SEQ ID NO. 5.

CTLA4-Ig alone or in combination with anti-CD 40L antibodies can prevent acute kidney allograft rejection (Kirk AD et al, 1997). In 1998, halbert et al, CTLA4-Ig and MR1 were used to suppress immunity, and AAV pulmonary re-administration was successfully achieved in mice (Halbert CL et al, 1998). Lorain et al, intramuscular injection of AAV1, delivered small nuclear RNA for the treatment of DMD. Exogenous genes can be expressed continuously when AAV1 is re-administered after mice are treated with CTLA4-Ig and MR 1. However, AAV1 re-injection requires repeated treatment of CTLA4-Ig and MR1 (Lorain et al, 2008). McIntosh et al demonstrated that after 6 injections of non-depleting anti-CD 4 (NDCD 4) antibodies and cyclosporin (CyA), the humoral immune response of the second AAV vector was inhibited by systemic injection and new transgene expression was not affected (McIntosh JH et al 2012).

In certain embodiments, the immunosuppressive protein (e.g., extracellular domain of CTLA-4 or fragment thereof, or extracellular domain of CD40 or fragment thereof, etc.) can be configured to be soluble. Thus, according to certain embodiments, the immunosuppressant protein encoded by the inducer nucleic acid vector can comprise a signal peptide.

Herein, the signal peptide is configured to allow secretion of the polypeptide outside the host cell once translated, and may be cleaved after secretion. The signal peptide ranges from 10 to 50 amino acid residues in length. According to certain embodiments, the signal peptide may have the sequence shown in SEQ ID NO. 6 or SEQ ID NO. 7. Other options for signal peptides may include any of the Oncoinhibin M signal peptide having the amino acid sequence of SEQ ID NO. 10 (see U.S. patent application publication No. 2003/0219863A 1), CD5 (Jones NH et al, 1986), or signal peptides from any other extracellular protein. It is noted that certain sequence variations, such as less than 10%, 20%, 30%, 40% or 50%, may exist for a signal peptide as described above, so long as the variant signal peptide retains signal peptide functionality.

In certain embodiments of the methods, the at least one immunosuppressive agent encoded by the inducer nucleic acid vector comprises at least one immunosuppressive nucleic acid, which may be DNA or RNA. In some embodiments, the inducer nucleic acid vector encodes a shRNA, siRNA or microrna that specifically targets one or more positive modulators in the immunostimulatory pathway as described above, such that expression of the positive modulator in the subject is down-regulated, resulting in an immune response to the inducer and the target agent being suppressed. In addition to the RNA interference mediated immunosuppression nucleic acids described above, other nucleic acids are also possible.

In certain embodiments, the at least one immunosuppressant provided herein may be used in combination with at least one additional immunosuppressant. Optionally, each immunosuppressant and each additional immunosuppressant may act on a different immunomodulator, and further preferably, these different modulators may be in the same or different signaling pathways (i.e., any of the immunostimulatory pathways or any of the immunosuppressive pathways) configured to have a synergistic effect to better suppress immune responses against the inducer and the target vehicle. Any suitable combination of immunosuppressants as disclosed in the present disclosure may be used. In certain embodiments, the at least one immunosuppressant and the at least one additional immunosuppressant inhibit binding or signaling of CD28 and CD80 (B7-1) and CD86 (B7-2), respectively, and inhibit binding or signaling of CD40 and CD40L (CD 154), respectively.

In certain embodiments, the combination of immunosuppressants can be delivered by one inducer vehicle. For example, an inducer vector can include an inducer nucleic acid vector encoding two or more immunosuppressants, which can be in one expression cassette or in different expression cassettes. For example, an inducer vehicle may include a combination of inducer nucleic acid vectors encoding two or more immunosuppressants, which may be in one expression cassette or in different expression cassettes.

Alternatively, in certain other embodiments, the combination of immunosuppressants may be delivered separately by a combination of inducer vehicles. For example, a first inducer vehicle can include a first inducer nucleic acid vector encoding a first immunosuppressant and a second inducer vehicle can include a second inducer nucleic acid vector encoding a second immunosuppressant. Where two or more inducer vehicles are used, each of the inducer vehicles is immunogenic and induces substantially the same immune response. For example, both the first inducer vehicle and the second inducer vehicle can be the same type of viral particle, and optionally can be the same serotype or share the same capsid protein.

In certain embodiments of the methods, step a) of the methods provided herein may further comprise: at least one additional inducer vehicle is administered to the subject. Each additional inducer nucleic acid vector herein can contain an inducer nucleic acid vector encoding at least one additional immunosuppressant.

In certain embodiments, the at least one immunosuppressant encoded by the inducer nucleic acid vector and the at least one additional immunosuppressant encoded by the additional inducer nucleic acid vector are configured to inhibit binding or signaling of CD28 and CD80 (B7-1) and CD86 (B7-2), respectively, and to inhibit binding or signaling of CD40 and CD40L (CD 154), respectively. Further optionally, an immunosuppressant comprises an extracellular domain of CTLA-4 or a fragment thereof capable of binding to at least one of CD80 (B7-1) or CD86 (B7-2) (e.g., CTLA 4-Ig); and/or an additional immunosuppressant comprises an extracellular domain of CD40 or a fragment thereof capable of binding to CD40L (CD 154) (e.g., CD 40-Ig).

immunosuppressant pharmaceutical preparation

In certain embodiments of the methods, step a) of the methods provided herein further comprises administering to the subject at least one immunosuppressive agent, each immunosuppressive agent capable of inhibiting or enhancing the activity of a target in an immunostimulatory pathway.

In certain embodiments, the immunosuppressive agent can be a compound, a nucleic acid, a polypeptide, a protein, or a combination thereof, and the immunosuppressive agent is not associated with an inducer vehicle. The immunosuppressive agent can be administered in combination with the inducer vehicle, e.g., before or after or simultaneously with the inducer vehicle, to induce an immunosuppressive effect on the inducer vehicle.

The immunosuppressive agent administered "in combination" with the inducer vehicle need not be administered simultaneously with or in the same composition as the inducer vehicle. As the phrase is used herein, an immunosuppressive agent administered before or after an inducer vehicle is considered to be administered "in combination" with the inducer vehicle, even though it is administered via a different route. Immunosuppressant agents administered in combination with the inducer vehicles disclosed herein are administered according to the schedules listed in the product information table of immunosuppressant agents or according to the Physicians 'Desk Reference 2003 (physician's Desk Reference, 57 th edition; medical economics company (Medical Economics Company); ISBN:1563634457; 57 th edition (11 2002)), or protocols well known in the art, where possible.

Herein, administration of an immunosuppressive agent may involve the use of certain vehicles, such as nanocarriers or nanoparticles, in which the immunosuppressive agent is encapsulated.

According to certain embodiments, the immunosuppressive agent may include a compound that may include any one of the following:

(1) Glucocorticoids such as prednisone (prednisone), prednisolone (prednisolone), methylprednisolone (Methylprednisolone), budesonide (budesonide), dexamethasone (dexamethasone), hydrocortisone (hydrocortisone), and the like;

(2) Purine analogs, such as azathioprine and mercaptopurine;

(3) Pyrimidine analogs such as fluorouracil and the like;

(4) Protein synthesis inhibitors;

(5) Folic acid analogs such as methotrexate and the like;

(6) Alkylating agents such as nitrogen mustard (cyclophosphamide), nitrosoureas, platinum compounds, and the like;

(7) Cytotoxic antibiotics such as dactinomycin (dactinomycin), anthracycline (anthracycline), mitomycin C (mitomycin C), bleomycin (bleomycin), mithramycin (mithramycin), and the like;

(8) Drugs acting on immunophilins (e.g., calcineurin, cyclophilin, FKBP 1A), such as calcineurin inhibitors (e.g., tacrolimus (tacrolimus), cyclosporine (cycloporine)), mTOR inhibitors (e.g., sirolimus (sirolimus) (i.e., rapamycin), everolimus), etc.;

(9) Janus kinase inhibitors such as tofacitinib (tofacitinib) and the like;

(10) IMDH inhibitors such as azathioprine, leflunomide (leflunomide), mycophenolate esters, and the like;

(11) Biopharmaceuticals, such as abapple (orexin (orecia)); beracep (Nulojix) noose Ji Ke; adalimumab (adalimumab) (sumira); anakinra (keneret); cetuximab (certolizumab) (simenda (Cimzia)); etanercept (Enbrel); golimumab (Xin Puni (simmoni)); infliximab (quasi-g (Remicade)); ikagroup mab (ixekizumab) (topology (Taltz)); natalizumab (natalizumab) (tasabari (Tysabri)); rituximab (rituximab) (Rituxan); secukinumab (Cosentyx); tolizumab (amerro (actera)); utekumab (Utekuumab) (Hida Nuo (Stelara)); vedolizumab (entiwei ao (Entyvio)); basiliximab (sully); daclizumab (daclizumab) (Jin Bulei tower (Zinbryta)).

In certain embodiments, the at least one immunosuppressive agent in the above embodiments of the method comprises a glucocorticoid, a calcineurin inhibitor, an mTOR inhibitor, a nitrogen mustard, a Janus kinase inhibitor, or a combination thereof.

In certain embodiments, the at least one immunosuppressive agent in the above-described embodiments of the method comprises tacrolimus, cyclosporine, cyclophosphamide, prednisone, prednisolone, methylprednisolone, budesonide, dexamethasone, hydrocortisone, rapamycin, everolimus, abamectin, beraprost, etanercept, or any combination thereof.

Herein, optionally, each of the at least one immunosuppressant and the at least one immunosuppressant agent acts on a different immunomodulator. In certain embodiments, the combination provides a synergistic effect in inhibiting a host immune response against the inducer vehicle and the target vehicle.

v. targeting agents

In any of the embodiments of the claims as described above, the targeting agent comprises a target nucleic acid vector encoding at least one target gene. The target gene as used herein is a transgene. The term "transgene" refers to an exogenous nucleic acid that encodes a gene product of interest and is introduced into a subject via a targeting agent. The gene product may be a natural biological molecule, for example, for treating a condition characterized by an deficiency in such natural molecule. The gene product may alternatively be non-natural, e.g., as an artificial product such as a mutant or fusion molecule, or as a naturally occurring product that is not naturally found in the subject. Biomolecules may be, for example, polypeptides/proteins and nucleic acids (e.g., polynucleotides, oligonucleotides).

In certain embodiments, the target gene may encode a biomolecule of interest or a functional fragment thereof. In certain embodiments, the target gene may encode a protein or peptide or biologically active fragment thereof.

In certain embodiments, the target gene may encode a nucleic acid (polynucleotide) of interest, such as a functional RNA. As used herein, a "functional RNA" may be an untranslated RNA, such as a nucleic acid sequence, that acts on a biological target, which modulates (increases or decreases) expression or activity (inhibits or enhances) of the biological target. For example, the functional RNA can be an antisense oligonucleotide, a ribozyme (e.g., as described in U.S. patent No. 5,877,022), an RNA that affects spliceosome-mediated/original splicing (see, e.g., puttaraju M et al, 1999; U.S. patent No. 6,013,487 and U.S. patent No. 6,083,702), an interfering RNA (RNAi), including small interfering RNAs (sirnas) that mediate gene silencing (see, e.g., sharp PA et al, 2000), micrornas or other non-translated functional RNAs, such as guide RNAs (see, e.g., gorman L et al, 1998 and U.S. patent No. 5,869,248), a single guide RNA used in CRISPR techniques (see, e.g., US10,266,850; US8,697,359; US20160298134 and Adli M et al, 2018), and the like.

In certain embodiments, the biomolecule of interest can be a therapeutic molecule (e.g., for medical or veterinary use). The term "therapeutic molecule" refers to a biological molecule (e.g., a polypeptide/protein or nucleic acid) that can be expressed in vivo to provide a therapeutic benefit.

Examples of therapeutic molecules include, but are not limited to, antibodies (e.g., monoclonal or bispecific or multispecific), insulin, glucagon-like peptide-1, peptide hormones, growth factors, erythropoietin (EPO), cytokines, extracellular domains of cell membrane proteins, clotting factors, antihemophilic factors, interferons, fc fusion proteins, and therapeutic enzymes (e.g., lysosomal hydrolases and sulfatases). Such therapeutic molecules also include nucleic acid molecules encoding such.

Examples of therapeutic molecules also include functional RNAs that target, for example, multiple Drug Resistance (MDR) protein targets, tumor targets (e.g., VEGF, HER2, EGFR, PD-L1, etc.), pathogen targets such as viral surface antigens (e.g., hepatitis b surface antigen genes), defective gene products (mutated dystrophin), or therapeutic targets as disclosed herein (e.g., myostatin).

Examples of therapeutic molecules include, but are not limited to, motor neuron survival gene 1 (SMN 1), alpha-N-acetylglucosaminidase (NAGLU), N-sulfoglucosaminesulfonyl hydrolase (SGSH), iduronate 2-sulfatase (IDS), coagulation Factor VIII (FVIII), coagulation Factor IX (FIX), bruton's tyrosine kinase (Bruton tyrosine kinase, BTK), ATP-binding cassette D subfamily member 1 (ABCD 1), acyl-coa dehydrogenase very long chain (ACADVL), androgen receptor repeat instability region (AR), hemoglobin subunit β (HBB), sodium voltage-gated channel alpha subunit 1 (SCN 1A), CF transmembrane conductance regulator (CFTR), colony stimulating factor 2 receptor subunit α (CSF 2 RA), interleukin 2 receptor subunit α (IL 2 AG), phenylalanine Hydroxylase (PHA), serine/threonine kinase 11 (STK 11), phosphatidylinositol glycan anchor biosynthesis class a (PIGA), ornithine carbamyl transferase (OTC), N-acetylglutamate synthase deficiency (NAGS), DM1 protein kinase (DMPK), CCHC type zinc finger nucleic acid protein (CNBP), acyl-coa dehydrogenase medium chain (ACADM), GNAS complex locus (as), fibrillin 1 (FBN 1), lipase a, gnpa, solute family member 7 (SLC 7 A7), the enzyme inhibitors comprise a hydroxyacyl-coa dehydrogenase trifunctional multi-enzyme complex subunit α (HADHA), a Growth Hormone Receptor (GHR), isovaleryl-coa dehydrogenase (IDV), alkaline phosphatase, biomineralization-related (ALPL), solute carrier family 25 member 15 (SLC 25A 15), huntingtin (huntingtin, HTT), carboxylated holoenzyme synthase (HCS), NOTCH receptor 3 (NOTCH 3), aldolase, fructose diphosphate B (ALDOB), atpase copper transfer β (ATP 7B), acid glucosidase α (GAA), glutaryl-coa dehydrogenase (GCDH), solute carrier family 12 member 3 (SLC 12 A3), glucosylceramidase β (GBA), familial mediterranean (Familial Mediterranean Fever, MEFV), galactosidase α (GLA), chloride voltage-gated channel 1 (CLCN 1), nuclear receptor subfamily 0 group B member 1 (NR 0B 1), argininosuccinase 1 (ASS 1), solute carrier family 25A13 (SLC 22 a), acetyl carrier family 22a (SLC 1), acetyl-coa 2B, and acetyl-coa 2 (asn-5 d).

In certain embodiments, the biomolecule of interest can be a prophylactic molecule (e.g., for a vaccine). In certain embodiments, the prophylactic molecule can be an immunogenic protein, a peptide, a nucleic acid encoding the immunogenic protein and peptide, or a functional RNA. The prophylactic molecule can be any biomolecule suitable for protecting a subject from diseases including, but not limited to, infectious diseases such as microbial, bacterial, protozoan, parasitic, fungal and viral diseases, as well as cancer. For example, the immunogen may be an orthomyxovirus immunogen (e.g., an influenza virus immunogen such as an influenza virus Hemagglutinin (HA) surface protein or an influenza virus nucleoprotein gene, or an equine influenza virus immunogen), or a lentivirus immunogen (e.g., an equine infectious anaemia virus immunogen, a Simian Immunodeficiency Virus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV) immunogen such as an HIV or SIV envelope GP160 protein, an HIV or SIV matrix/capsid protein, and HIV or SIV gag, pol, and env gene products). The immunogen may also be an arenavirus immunogen (e.g., an arenavirus (Lassa fever virus) immunogen, such as an arenavirus nucleocapsid protein gene and an arenavirus envelope glycoprotein gene), a poxvirus immunogen (e.g., a vaccinia such as a vaccinia Ll or L8 gene), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus) immunogen or a Marburg virus (Marburg virus) immunogen, such as NP and GP genes), a bunyavirus immunogen (e.g., an RVFV, CCHF and SFS virus) or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as a human coronavirus envelope glycoprotein gene, or a swine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen, or a Severe Acute Respiratory Syndrome (SARS) immunogen, such as S (Sl or S2), M, E or N protein or an immunogenic fragment thereof, or a covd-19 immunogen. The immunogen may further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis a, hepatitis b or hepatitis c) immunogen, or any other vaccine immunogen known in the art. For another example, the immunogen may be a tumor or cancer antigen expressed on the surface of tumor or cancer cells. Exemplary tumor or cancer antigens include, but are not limited to, b-catenin, BRCA1 gene product, BRCA2 gene product, epCAM, EGFR, her2, VEGFR, CD19, PSMA, and the like.

In certain embodiments, the biomolecule of interest may be a biomolecule of interest for experimentation or research, such as a reporter protein (e.g., for animal models) and a nuclease (e.g., for genetic engineering purposes), and the like.

The reporter protein can be expressed in an animal (e.g., mouse, rat, guinea pig, etc.) to provide an engineered animal model. Examples of reporter proteins include, but are not limited to, fluorescent proteins (e.g., EGFP, GFP, RFP, BFP, YFP or dsRED 2), enzymes that produce detectable products, such as luciferases (e.g., from gaussian, renilla or Pho), b-galactosidase, b-glucuronidase, alkaline phosphatase, and chloramphenicol acetyl transferase genes, or proteins that can be detected directly. Almost any protein can be detected directly by using, for example, specific antibodies to the protein. Additional markers (and related antibiotics) suitable for either positive or negative selection of eukaryotic cells are disclosed in Sambrook and Russell,2001 and Ausubel FM et al, 1992, including periodic updates.

Nucleases can be useful for in vivo gene editing. Examples of nucleases include, but are not limited to, zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or Cas family proteins (such as Cas1, cas1B, cas, cas3, cas4, cas5, cas6, cas7, cas8, cas9 (also referred to as Csn1 and Csx 12), cas 10, cas 11, cas12, cas13, csy1, csy2, csy3, cse1, cse2, csc1, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmr1, cmr3, cmr4, cmr5, cmr6, csb1, csb2, csb3, csx17, csx14, csx10, csx16, csaX 3, csx1, csx15, csf1, csf2, f3, csf4 or Cpf 1).

Nucleic acid vectors

In some embodiments, the inducer vector comprises an inducer nucleic acid vector encoding the at least one immunosuppressant.

In some embodiments, the targeting agent comprises a target nucleic acid vector encoding the at least one target gene.

Herein, a vector may comprise an exogenous nucleic acid, typically in the form of a polynucleotide construct, and used to deliver to a host at least one transgene encoding a certain polypeptide or a certain polynucleotide (RNA or DNA).

Vectors typically include regulatory elements operably linked to a coding sequence (e.g., a transgene) to effect and regulate expression thereof. As used herein, the term "operably linked" means that the coding sequence is directly or indirectly linked or associated with one or more regulatory sequences in an exogenous nucleic acid in a manner that allows expression of the protein of interest from the coding sequence in the cell. The coding sequence, together with the regulatory sequences, may be referred to herein as an expression cassette. In certain embodiments, the exogenous nucleic acid can be in the form of an expression vector. Examples of transcriptional regulatory elements include one or more promoters and/or enhancers, and optionally polyadenylation sequences and/or one or more introns interposed between exons of the protein coding sequence. As used herein, the terms "regulatory element," "regulatory sequence," and the like refer to any nucleotide sequence that is necessary or advantageous for expression of a coding sequence. Regulatory sequences may include, but are not limited to, one or more promoters, enhancers, transcription terminators, polyadenylation sequences, signal peptide coding sequences, internal ribosome entry sites, and/or one or more introns interposed between exons of a protein coding sequence.

Virus vector

In some embodiments, the nucleic acid vectors provided herein include viral vectors. As used herein, "viral vector" refers to a single-stranded or double-stranded nucleic acid vector having a 5 'viral terminal repeat and/or a 3' viral terminal repeat at the 5 'and/or 3' end of a nucleic acid sequence of interest (e.g., an expression construct encoding a protein of interest). The viral vector may comprise a pair of TRs or a single TR. The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and mediates a desired function such as replication, viral packaging, integration, and/or proviral rescue. For example, the 5 'viral terminal repeat sequence and the 3' viral terminal repeat sequence may contain an origin of replication and allow initiation of DNA synthesis at one viral terminal repeat and continued to the other viral terminal repeat. Examples of viral terminal repeats include, but are not limited to, inverted Terminal Repeats (ITRs) (e.g., those included in adeno-associated viruses (AAV)), long Terminal Repeats (LTRs) (e.g., those included in retroviruses), and the like. The viral vector may include one or more sequences heterologous to the viral genome between ITRs.

Viral vectors may be prepared using methods known to those of ordinary skill in the art or otherwise described herein. For example, viral vectors may be constructed and/or purified using methods such as those shown in U.S. Pat. No. 4,797,368 and Laughlin CA et al, 1983.

The viral vector may be packaged in a viral particle. A variety of viral vectors are known in the art to be suitable for delivering nucleic acids to a subject such as a human. The most commonly used viral vectors include those derived from adenoviruses, adeno-associated viruses (AAV) and retroviruses, including lentiviruses, such as Human Immunodeficiency Virus (HIV). Retroviral vectors, adenoviruses and AAV provide an effective and useful method for the efficient introduction and expression of exogenous genes in mammalian cells. These vectors have a broad host and cell type range and stably and efficiently express genes. The safety of these vectors is well understood in the art. Other viral vectors that may be used to transfer genes into a subject include the herpes virus papovaviruses, such as JC, SV40, polyomaviruses; epstein-Barr Virus (EBV); papillomaviruses such as bovine papilloma virus type I (BPV); polioviruses and other human and animal viruses.

More detailed descriptions of viral vectors can be found in other parts of the disclosure, particularly in subsequent parts, which encompass various types of viral vectors that can be generally used as gene delivery vehicles for subjects.

a) AAV vectors

In certain embodiments, the nucleic acid vectors provided herein include related viral (AAV) vectors. AAV is a single-stranded human DNA parvovirus of about 4.7kb in genome size. AAV genomes contain two major genes: rep genes encoding Rep proteins (Rep 76, rep 68, rep 52, and Rep 40); and cap genes encoding AAV structural proteins (VP-1, VP-2, and VP-3), flanked by 5 'Inverted Terminal Repeats (ITRs) and 3' ITRs. As used herein, the term "AAV vector" encompasses any viral vector comprising one or more heterologous sequences flanking at least one or two AAV inverted terminal repeat sequences. As is well understood in the art, the term "AAV ITR" is a sequence of about 145 nucleotides present at both ends of the native single stranded AAV genome. The 125 nucleotides of the outermost layer of the ITR can exist in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions, allowing intra-strand base pairing to occur within this portion of the ITR.

AAV ITRs can be derived from any AAV, including but not limited to AAV serotype 1 (AAV 1), AAV2, AAV3, AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, AAV 11, AAV 12, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV, and any other AAV now known or later discovered (see, e.g., bernard NF et al, 2007, and Gao G et al, 2004). The nucleotide sequence of the AAV ITR region is known. (see, e.g., kotin RM,1994 and Berns KI, 1990). Early description of AAV1, AAV2, and AAV3 terminal repeats can be found in Xiao, x.,1996, which is incorporated herein in its entirety.

AAV ITRs can be native AAV ITRs, or alternatively can be altered from native AAV ITRs, e.g., by mutation, deletion, or insertion, so long as the altered ITRs can still mediate desired biological functions, such as replication, viral packaging, integration, and the like. The 5 'and 3' itrs flanking the selected nucleotide sequence in the AAV vector need not be identical or derived from the same AAV serotype, so long as they function as intended, e.g., allowing excision and rescue of the sequence of interest and integration into the recipient cell genome. The genomic sequence of AAV and AAV rep and cap genes are known in the art and can be found in literature and public databases such as the database of gene banks (GenBank database).

In some embodiments, the AAV vector may be recombinant. Recombinant AAV vectors may comprise one or more heterologous sequences of different viral origin (e.g., sequences from non-AAV viruses, or from AAV of different serotypes, or from partial or complete synthesis). In certain embodiments, the heterologous sequence flanks at least one AAV ITR.

In certain embodiments, the AAV vectors provided herein have a size suitable for packaging into AAV viral particles. For example, the size of an AAV vector may reach a size limit of the genome size of the AAV to be used, e.g., up to 5.2kb. In certain embodiments, the AAV vector is no more than 5.2 kilobases (kb) in size, no more than about 5kb, no more than about 4.5kb, no more than about 4kb, no more than about 3.5kb, no more than about 3kb in size, no more than about 2.5kb in size, see, e.g., dong JY et al, 1996.

Due to packaging size limitations of individual AAV, two or more AAV vectors may be constructed in a manner that allows reconstitution of the complete sequence or expression cassette in cells co-transfected with these AAV vectors for heterologous sequences that exceed the packaging capabilities of the individual AAV vectors. Methods for constructing such AAV vectors are known in the art, e.g., for constructing overlapping binary vectors, trans-splicing vector pairs, hybrid vector systems, and more detailed information is available in Chamberlain K et al, 2016 and U.S. patent No. 6,596,535.

b) Adenovirus vector

In certain embodiments, the exogenous nucleic acid comprises an adenovirus vector. Adenoviruses have a double-stranded linear DNA genome that cannot integrate into the host genome. Illustrative examples of adenovirus vectors include first generation adenovirus vectors (e.g., adenovirus vectors lacking the E1a gene and the E1b gene, and adenovirus vectors lacking the E1 gene and the E3 gene), second generation adenovirus vectors (e.g., adenovirus vectors lacking the E1 gene and the E2 gene, adenovirus vectors lacking the E1 gene and the E4 gene), and enteroless adenovirus vectors in which all viral coding sequences are deleted (also known as helper-dependent adenovirus vectors).

Typically, to work with adenovirus vectors, certain replication-defective adenovirus vectors are produced in a complementing cell line configured to provide at appropriate levels gene functions not present in the replication-defective adenovirus vector but required for viral propagation, in order to produce high titres of viral transfer vector stock. The complementing cell line may complement the defect in at least one replication-essential gene function encoded by the early region, the late region, the virus packaging region, the virus-associated RNA region, or a combination thereof, including all adenovirus functions (e.g., enabling propagation of an adenovirus amplicon). Construction of the complementing cell lines involves standard molecular biology and cell culture techniques known to those of ordinary skill in the art, such as those described in Sambrook et al, 1989 and Ausubel FM et al, 1994.

Supplementary cell lines for the production of adenovirus vectors include, but are not limited to, 293 cells (described, for example, in Graham FL et al 1977), PER.C6 cells (described, for example, in International patent application WO 97/00326 and U.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (described, for example, in International patent application WO 95/34671 and Brough DE et al 1997). In some cases, the complementing cells will not complement all of the necessary adenovirus gene functions. Helper viruses may be used to provide trans-gene functions not encoded by the cell or adenovirus genome to effect replication of the adenovirus vector. Adenovirus vectors can be constructed, propagated, and/or purified using, for example, the materials and methods described below: U.S. Pat. nos. 5,965,358, 6,033,908, 6,329,200, 6,383,795, 6,447,995 and 6,475,757; U.S. patent application publication No. 2002/0034735A1 and International patent applications WO 98/53087, WO 98/56937, WO 99/15686, WO 99/54441, WO 00/12765, WO 01/77304 and WO 02/29388, among others identified herein. non-C group adenovirus vectors, including adenovirus serotype 35 vectors, may be generated using, for example, the methods described in U.S. Pat. Nos. 5,837,511 and 5,849,561, international patent applications WO 97/12986 and WO 98/53087.

c) Lentiviral vector

In certain embodiments, the nucleic acid vectors provided herein include lentiviral vectors. Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol and env, other genes with regulatory or structural functions. Illustrative examples of lentiviral vectors include, but are not limited to, vectors derived from HIV-1, SIV, FIV, CAEV, VMV and EIAV.

Lentiviral vectors may be generated using any method known in the art. Examples of lentiviral vectors and/or methods of their production can be found, for example, in U.S. publication nos. 20150224209, 20150203870, 20140335607, 20140248306, 20090148936, and 20080254008, which are incorporated herein by reference.

As an example, when the lentiviral vector is not capable of integration, the lentiviral genome further comprises an origin of replication (ori), the sequence of which depends on the nature of the cell in which the lentiviral genome must be expressed. The origin of replication may be from eukaryotic, optionally mammalian, and further optionally human sources. Since the lentiviral genome does not integrate into the cell host genome (because of defective integrase), the lentiviral genome may be lost in cells that undergo frequent cell division; this is especially the case in immune cells such as B or T cells. In some cases, the presence of an origin of replication may be beneficial. The vector particles may be produced after transfection of suitable cells such as 293T cells by the plasmid or by other methods. In cells used to express lentiviral particles, all or some of the plasmids may be used to stably express their encoding polynucleotides or transiently or semi-stably express their encoding polynucleotides.

d) Retroviral vectors

In certain embodiments, the nucleic acid vectors provided herein include retroviral vectors. Retroviruses have an RNA genome and can replicate in host cells by reverse transcriptase to produce DNA from the RNA genome. Illustrative examples of retroviral vectors include, but are not limited to, vectors derived from: avian leukemia virus, mouse mammary tumor virus, murine leukemia virus, bovine leukemia virus, micropterus salmoides skin sarcoma virus (Walleye dermal sarcoma virus), HIV-1 (human immunodeficiency virus), HIV-2, SIV (simian immunodeficiency virus), EIAV (equine infectious anemia virus), FIV (feline immunodeficiency virus), CAEV (caprine arthritis encephalitis virus), VMV (visna/maedi virus)), human foamy virus, moloney murine leukemia virus (moloney murine leukaemia virus), rous sarcoma virus (rous sarcoma virus), feline leukemia virus, human T lymphocyte virus, and simian foamy virus.

e) Other viral vectors

Methods for producing other viral vectors as provided herein are known in the art and may be similar to the methods exemplified above.

Preparation of vehicle

Inducer vehicles and target vehicles can be prepared using methods known in the art.

In certain embodiments, the inducer mediator and/or the target mediator is a viral mediator. The viral vector may comprise a viral vector packaged within a virosome.

Viral vectors can be constructed using methods known in the art. The general principles of recombinant viral vector construction are known in the art. See, for example, carter BJ et al, 1992 and Muzyczka N et al 1992. For example, a heterologous sequence may be inserted directly between Terminal Repeats (TRs) of a viral genome (e.g., AAV), wherein some unwanted viral genes have been deleted. Other parts of the viral genome may also be deleted, provided that sufficient TR parts remain to allow replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Pat. nos. 5,173,414 and 5,139,941; international publication Nos. WO 92/01070 and WO 93/03769; lebkowski JS et al 1988; vincent KA et al 1990; carter BJ et al, 1992; muzyczka N et al 1992; kotinRM, 1994; shelling and Smith,1994; and Zhou SZ et al, 1994.

Alternatively, viral TR may be excised from the viral genome or from a viral vector containing the viral genome and fused to 5 'and 3' of the heterologous sequence using standard ligation techniques such as those described in Sambrook J et al, 1989. AAV vectors containing AAV ITRs are commercially available and have been described, for example, in U.S. patent No. 5,139,941.

In certain embodiments, the viral vector comprises a recombinant viral particle. The viral particles may be produced from a viral expression vector. Viral particles may be produced by introducing the viral expression vector into a suitable host cell using known techniques, such as by transfection, along with other necessary mechanisms such as plasmids encoding certain viral genes (e.g., AAV cap/rep genes) and helper genes provided by adenovirus or herpes viruses (see, e.g., naso MF et al, 2017, incorporated herein in its entirety). Viral expression vectors can be expressed in host cells and packaged into viral particles.

In some embodiments, the viral vector further comprises a capsid protein. In some embodiments, the capsid protein may be native or recombinant. In some embodiments, the capsid protein may be modified or chimeric or synthetic. The modified capsid may comprise modifications such as insertions, additions, deletions or mutations. For example, the modified capsid may incorporate a detection or purification tag. The chimeric capsid comprises portions of two or more capsid sequences. Synthetic capsids include sequences that are synthetic or designed artificially.

In some embodiments, the viral vector comprises an AAV vector. The capsid structure of AAV is also known in the art and is described in more detail in Bernard NF et al, 2007. In some embodiments, the capsid proteins are derived from two or more AAV serotypes. It is known in the art that various AAV serotypes are functionally and structurally related, even at the genetic level (see, e.g., blacklow NR,1988 and Rose J, 1974). However, AAV viral particles of different serotypes may have different tissue tropisms (see non macher M et al 2012 for details), and gene therapy for target tissue may be appropriately selected. In some embodiments, the cap gene or the capsid protein may have specific tropism properties. The term "tropism property" refers to the transduction pattern of one or more target cells, tissues and/or organs. For example, the capsid protein may have a tropism property specific to the liver (e.g., hepatocytes), brain, eye, muscle, lung, kidney, intestine, pancreas, salivary gland, or synovium, or any other suitable cell, tissue, or organ.

In some embodiments, the cap gene or the capsid protein is derived from any suitable AAV capsid gene or protein, such as, but not limited to, AAV capsid genes or proteins derived from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV843, AAVbb2, AAVcys, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64, AAVhu37, AAV3B, AAVhu48, AAVhu43, AAVhu44, AAVhu46, AAVhu19, AAVhu20, AAVhu23, AAVhu22, AAVhu24, AAVhu21, AAVhu27, AAVhu28, AAVhu29 AAVhu63, AAVhu64, AAVhu13, AAVhu56, AAVhu57, AAVhu49, AAVhu58, AAVhu34, AAVhu45, AAVhu47, AAVhu51, AAVhu52, AAVhu T41, AAVhu S17, AAVhu T88, AAVhu T71, AAVhu T70, AAVhu T40, AAVhu T32, AAVhu T17, AAVhu LG15, AAVhu9, AAVhu10, AAVhu11, AAVhu53, AAVhu55, AAVhu54 strains of AAVhu7, AAVhu18, AAVhu15, AAVhu16, AAVhu25, AAVhu60, AAVch5, AAVhu3, AAVhu1, AAVhu4, AAVhu2, AAVhu61, AAVrh62, AAVrh48, AAVrh54, AAVrh55, AAVcy2, AAVrh35, AAVrh37, AAVrh36, AAVcy6, AAVcy4, AAVcy3, AAVcy5, AAVrh13, AAVrh38, AAVhu66, AAVhu42, AAVhu67, AAVhu40, AAVhu41, AAVrh40, AAVrh2, AAVbb1, AAVhu17, AAVhu6, AAVrh25, aavri 2, aavri 3, AAVrh57, AAVrh50, vrh49, vrh39, AAVrh61, AAVrh 31, AAVrh-35, AAVrh32, AAVrh 31, AAVrh-35, or the like. The capsid of AAV843 is identical to the synthetic capsid AAVXL32 disclosed in WO 2019241324A1 (which is incorporated herein in its entirety), and AAV843 is also disclosed in, for example, xu j. Et al, 2019.

Further examples of AAV capsid gene sequences and protein sequences can be found in the database of gene banks, see gene bank accession numbers: AF043303, AF, J02275, J01901, J02275, X01457, AF, AH, AY, NC, AF, AY, NC AY, AF085716, AF, AY, AAS, AY243015, AY, AX, AY, AX, AY, AF085716, AF, AY, AAS, AY243015, and AY, AY AY, AY243020, AY243000, AY, AY_243020, AY_243000, AY_20, AY_2420, AY_ AY, NC, AY, NC 001829, AY, NC 001829, AY, AX, AY, NC, AY, NC, AY, AX NC 001829, AY.

In certain embodiments, the AAV vector comprises cap genes from one AAV serotype and AAV ITRs from a second serotype. In certain embodiments, the AAV vector comprises an AAV viral particle comprising a pseudotyped AAV. "pseudotyped" AAV refers to AAV that contains a viral genome that comprises a capsid protein from one serotype and a 5'-3' ITR comprising a second serotype. Pseudotyped AAV would be expected to have cell surface binding properties of the capsid protein-derived serotype, as well as genetic properties consistent with the ITR-derived serotype.

Any recombinant method can be used to produce AAV particles or virions that include the transgene of interest. AAV-producing cells, AAV packaging cells, and packaging techniques known to those of skill in the art (see, e.g., U.S. patent No. 5,436,146; 5,753,500, 6,040,183, 6,093,570, and 6,548,286, and US 2002/0168342).

Generally, the methods involve culturing packaging cells containing a nucleic acid sequence encoding an AAV capsid protein or fragment thereof; a functional rep gene; a recombinant AAV vector comprising an AAV Inverted Terminal Repeat (ITR) and a transgene; and sufficient helper functions to allow packaging of the recombinant AAV vector into AAV capsid proteins. The components to be cultured in the host cell to encapsulate the rAAV vector in the AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the desired components (e.g., recombinant AAV vectors, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell that has been engineered to contain one or more of the desired components using methods known to those of skill in the art. Most suitably, such stable host cells may contain the desired components under the control of an inducible promoter. However, the desired components may be under the control of a constitutive promoter. Any suitable genetic element may be used to deliver the recombinant AAV vectors, rep sequences, cap sequences, and ancillary functions required for the production of the rAAV of the invention to packaging host cells. The selected genetic elements may be delivered by any suitable method, including the methods described herein. Methods for constructing any of the embodiments of the present invention are known to the nucleic acid operator and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., sambrook J et al, 1989. Similarly, methods of producing rAAV virions are well known and the selection of suitable methods is not a limitation of the present invention. See, for example, fisher KJ et al, 1993 and U.S. Pat. No. 5,478,745.

Any producer cell used in the art to produce rAAV viral particles can be used to produce the subject vectors, including, for example, mammalian cells, insect cells, microorganisms, yeast, and the like.

In one example, a recombinant AAV (rAAV) vector comprising a transgene can be transfected into a mammalian producer cell using the mammalian producer cell. In some embodiments, recombinant AAV particles can be produced using triple transfection (see, e.g., us patent No. 6,001,650). Typically, recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (including a transgene) to be packaged into AAV particles, AAV helper function vectors, and helper function (accessory function) vectors. Typically, AAV helper function vectors encode AAV helper function sequences (rep and cap) that act in trans on productive AAV replication and encapsidation. Optionally, the AAV helper function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Helper function vectors may encode nucleotide sequences that are not AAV-derived viruses and/or cellular functions upon which AAV replicates. Helper functions include functions required for AAV replication, including but not limited to those involving activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. The viral-based accessory function may be derived from any known helper virus, such as adenovirus, herpes virus (other than herpes simplex virus type 1) and vaccinia virus. In some cases, packaging cells may be used in which AAV rep and cap genes are stably maintained in a host cell (e.g., 293 cells or Hela cells). In some cases, the host cell is a producer cell in which the AAV vector genome is stably maintained and packaged. The producer cells can be cultured into producer rAAV particles that are purified and formulated using standard techniques known in the art.

In another example, an AAV expression vector may be packaged as a baculovirus and introduced into an insect-producing cell (e.g., sf9 cell). Also introduced into insect cells by another baculovirus are AAV REP and CAP genes. Baculovirus, as a virus, includes genes encoding helper functions necessary for efficient rAAV virus production. Thus, upon infection of insect cells by two baculoviruses, the producer cells can be cultured to produce rAAV, and AAV vectors purified and formulated using standard techniques known in the art.

In certain embodiments, the inducer mediator and/or target mediator is a non-viral mediator. In some embodiments, the non-viral inducer vehicle comprises an immunogenic synthetic nanocarrier capable of delivering the at least one immunosuppressant or a nucleic acid vector encoding the at least one immunosuppressant.

In some embodiments, the targeting agent comprises an immunogenic synthetic nanocarrier capable of delivering the at least one target gene or a nucleic acid vector capable of delivering the at least one target gene.

In other words, delivery of a vehicle to a subject does not require a viral particle comprising a nucleic acid vector, involving delivery of a nucleic acid vector using a non-viral vector.

Synthetic nanocarriers can be prepared using a variety of methods known in the art. Such references include U.S. patent nos. 5,578,325 and 6,007,845 and Doubrow,1992; mathiowitz E et al, 1987a, mathiowitz E et al, 1987b, mathiowitz E et al, 1988 and Paolicelli P et al, 2010.

For example, the synthetic nanocarriers can be formed by the following methods: such as nano-precipitation, flow focusing using fluid channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, micro-emulsion procedures, precision machining, nano-fabrication, sacrificial layers, simple and complex coacervation, and other methods well known to those of ordinary skill in the art. Alternatively, aqueous and organic solvent syntheses for monodisperse semiconductor nanomaterials, conductive nanomaterials, magnetic nanomaterials, organic nanomaterials, and other nanomaterials have been described in published U.S. patent nos. 5578325 and 6007845.

Various methods (including but not limited to published literature) can be used to encapsulate various materials into synthetic nanocarriers as desired (e.g., astete CE et al, 2006;Avgoustakis K,2004; and Reis CP et al, 2006; and Paolicelli P et al, 2010). Other methods suitable for encapsulating the material into synthetic nanocarriers may be used, including but not limited to the methods disclosed in U.S. patent No. 6,632,671.

In certain embodiments, the synthetic nanocarriers are prepared by a nano-precipitation method or spray drying. The conditions used to prepare the synthetic nanocarriers can be varied to produce particles having a desired size or property (e.g., hydrophobicity, hydrophilicity, external morphology, "tackiness," shape, etc.). The method of preparing the synthetic nanocarriers and the conditions (e.g., solvent, temperature, concentration, air flow rate, etc.) used may depend on the composition of the material and/or polymer matrix to which the synthetic nanocarriers are to be attached. If synthetic nanocarriers prepared by any of the methods described above have a size range that is outside of the desired range, such synthetic nanocarriers can be sized, for example, using a sieve.

The elements (i.e., components) of the synthetic nanocarriers may be attached to the entire synthetic nanocarriers, for example, by one or more covalent bonds, or may be attached by one or more linkers (e.g., amide linkers, disulfide linkers, thioether linkers, hydrazone linkers, hydrazide linkers, imine or oxime linkers, urea or thiourea linkers, amidine linkers, amine linkers, sulfonamide linkers, etc.). The compositions and methods may be adapted from published documents including Sharpless VV et al, 2002 and Meldal M et al, 2008, U.S. patent application Ser. No. 20060002852, 20090028910, international patent application Ser. No. WO 2008127532.

Alternatively or additionally, the synthetic nanocarriers may be directly or indirectly attached to the component via non-covalent interactions. In non-covalent embodiments, the non-covalent coupling is mediated by non-covalent interactions, including, but not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der waals interactions (van der Waals interaction), magnetic interactions, electrostatic interactions, dipole-dipole interactions, and/or combinations thereof. Such coupling may be disposed on the outer or inner surface of the synthetic nanocarriers. In embodiments, encapsulation and/or absorption is a form of coupling. In embodiments, the synthetic nanocarriers may be combined with therapeutic macromolecules or other compositions by mixing in the same vehicle or delivery system.

Application of

As used herein and in the broadest sense, the term "administration" refers to the administration or dispensing of a pharmaceutically active material to a subject in a pharmacologically compatible manner. The term is intended to include "causing to be administered," which means directly or indirectly causing, prompting, encouraging, helping, inducing or directing the administration of a pharmaceutically active material to another subject. It is noted that in some embodiments, the terms may also be exchanged for "delivery (deliver, delivery or delivery)", and the like. As used herein, a pharmaceutically active material includes a vehicle (i.e., an inducer vehicle and/or a target vehicle).

In a general sense, administration of the inducer and/or target vehicle can take a variety of routes, which can be systemic or in a localized treatment area. Optionally, the subject may be administered via parenteral, oral, enteral, intraoral, nasal, topical, rectal, vaginal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary, cardiac, subcutaneous, intraparenchymal, intraventricular, or intrathecal routes of administration. In certain embodiments, the inducer and/or target vehicle is delivered to the subject orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intrathecally, intraventricularly (ICV), or intrarectally. The route of administration may also include administration by inhalation or pulmonary aerosol. Techniques for preparing aerosol delivery systems are well known to those skilled in the art.

In certain embodiments, the vehicle comprises a viral particle (or viral particle), i.e., the inducer vehicle comprises an inducer viral particle, and/or the target vehicle comprises a target viral particle. Thus, methods and uses for administering or delivering viral particles include any mode compatible with a subject. In particular embodiments where the viral particles comprise AAV viral particles, the viral particles are administered to the subject intravenously, intra-arterially, intramuscularly, subcutaneously, orally, by cannula, via catheter, transdermally, intracranially, via inhalation, endoluminal, or mucosally.

In certain embodiments, the subject is additionally administered at least one immunosuppressive agent (e.g., a compound, nucleic acid, polypeptide, or combination thereof having immunosuppressive function) in combination with administration of a vehicle (e.g., an inducer vehicle). Herein, administration of the at least one immunosuppressive agent may involve the use of certain vehicles, such as nanocarriers or nanoparticles, in which the immunosuppressive agent is encapsulated. Furthermore, the at least one immunosuppressive agent need not be administered simultaneously with or in the same composition as the vehicle. In other words, the at least one immunosuppressive agent may be administered before or after the vehicle is administered, as desired, and may be administered via the same or a different route/manner as the vehicle.

Depending on the actual need, administration of a pharmaceutically active material (i.e., vehicle) may include only a single administration or include repeated administrations, i.e., more than one administration of the same pharmaceutically active material. As used herein, the term "repeated administration (repeated administration, repeatedly administer or repeatedly administering)" and the like refers to enhancing or extending a previously established effect (i.e., expression of a target gene or treatment of a condition, etc.) intended to be achieved by multiple administrations of a target agent in a subject. These embodiments generally relate to the (n+1) th administration of the target agent after the nth administration (n is an integer not less than 0), and the (n+1) th administration is generally performed when the effect on the establishment of the target agent at the nth administration is decreasing or is about to decrease.

For example, depending on whether the level of immune tolerance to an immunogenic target agent can be achieved in a subject, the inducer agent can be administered once and, if so, only once, or more than once (e.g., two, three, four, etc.) if subsequent administrations are required after the first administration to enhance the effect. In the latter case, repeated administration may be considered as being interchangeable with "repeated doses" or "repeated administrations" meaning that at least one additional dose or administration is administered to the subject following an early dose or administration of the same material. For example, the repeated dose of the viral particle is at least one additional dose of the viral particle following the previous dose of the same viral particle, and herein the viral particle may be any of an inducer viral particle or a target viral particle.

While the pharmaceutically active material used for repeated administration may be the same, the amount of material in repeated doses (i.e., the dose) may be the same as or different from the earlier dose. For example, in certain embodiments in which the inducer vehicle includes inducer viral particles, the amount of inducer viral particles in repeated doses (i.e., subsequently administered) may be less than the amount of inducer viral particles of earlier doses. Alternatively, the amount of repeat dose may be at least equal to the amount of inducer viral particles in the early dose.

In addition to dosages, the frequency of repeated administration or repeated administration of the pharmaceutically active material and/or the route/mode of administration may vary depending on the actual need. For example, repeated doses may be administered weeks, months or years after the previous dose. In some embodiments, the repeated dose or administration is administered at least 1 week after the dose or administration that occurs just prior to the repeated dose or administration.

Immune tolerance

The methods provided herein can induce immune tolerance to an immunogenic target agent in a subject by administering to the subject an immunogenic inducer agent as provided herein, e.g., an immunogenic inducer agent comprising an inducer nucleic acid vector encoding at least one immunosuppressant.

The immune tolerance can be determined, for example, by the antibody titer of anti-mediator antibodies in the serum of the subject. As used herein, the term "anti-mediator antibody" means one or more antibodies that can specifically bind to an inducer mediator and/or a target mediator.

In certain embodiments, the immune tolerance is characterized by the serum of the subject having an antibody titer of anti-mediator antibodies below a predetermined threshold level after administration of the immunogenicity-inducing agent. Antibody titers of anti-mediator antibodies can be measured by any suitable method known in the art, e.g., an ELISA assay, and can be determined by the dilution fold of serum to produce a threshold specific binding to an inducer mediator or target mediator, optionally present in a given amount. Depending on the different sensitivity requirements, different dilution factor thresholds may be selected to determine whether or not there is induced immune tolerance.

In certain embodiments, the immune tolerance is characterized by an antibody titer of the anti-mediator antibody of the serum of the subject after administration of the immunogenicity-inducing agent vehicle of no more than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200, and wherein the antibody titer is defined as the dilution factor by which the serum of the subject produces a threshold specific binding to the inducing agent vehicle or the target particle. In certain embodiments, specific binding of the anti-mediator antibody to the inducer mediator or target particles is determined by ELISA.

In some embodiments, threshold specific binding is determined based on a given amount of vehicle, wherein threshold specific binding is considered sufficient to show a level of antibody binding to the vehicle specific binding. In some embodiments, the binding is determined based on the following formula: (bonding) _{(presence of a vehicle)} -binding _{(absence of vehicle)} ) Combination of _{(absence of vehicle)} Wherein the threshold for specific binding can be set appropriately by one of ordinary skill in the art. In certain embodiments, the threshold for specific binding is set to 2, 2.5, 3, 3.5, 4, or 5.

In certain embodiments, the inducer mediator and/or target mediator is a viral particle, and the immune tolerance can be determined by the antibody titer of neutralizing antibodies in the serum of the subject. As used herein, the term "neutralizing antibody" means an antibody that can effectively inhibit viral particles from infecting their susceptible host cells. Neutralizing antibody titers can be analyzed by assessing the ability of serum antibodies to inhibit transduction or infection of a susceptible host cell (i.e., HUH-7 cells or HEK293 cells directed against AAV) by a viral particle of interest, which may express a reporter gene such as β -galactosidase or luciferase following transduction or infection. Neutralizing antibody titers can be determined by the dilution factor of serum to produce a maximum neutralization of about 50% of the inducer vehicle and/or target vehicle, optionally present in a given amount. Depending on the different sensitivity requirements, different dilution factor thresholds may be selected to determine whether or not there is induced immune tolerance.

In certain embodiments, the immune tolerance is characterized by an antibody titer of no more than 2, 4, 6, 8, 18, or 32 of neutralizing antibodies in the serum of the subject after administration of the immunogenicity inducer vehicle, and wherein the antibody titer is defined as the dilution ratio of the serum of the subject that produces 50% maximum neutralization with the inducer vehicle or the target vehicle.

Preexisting immunity

In certain embodiments, the subject has no pre-existing immune response to the inducer vehicle or the target vehicle or both.

As used herein, the phrase "pre-existing immune response" means an acquired immune response that exists prior to administration of an inducer vehicle to a subject. Such pre-existing immune responses may be obtained as a result of prior exposure to the relevant antigen. For example, if the inducer vehicle comprises AAV viral particles, prior exposure to or infection with the relevant AAV may result in an acquired immune response to the inducer vehicle even prior to administration of the inducer vehicle to the subject.

In certain embodiments, the pre-existing immune response is characterized by the serum of the subject having an antibody titer of anti-mediator antibodies above a predetermined threshold level prior to administration of the immunogenicity-inducing agent mediator. Similarly, the antibody titer of an anti-vehicle antibody can be measured by any suitable method known in the art, e.g., an ELISA assay, and different antibody titer thresholds can be selected to determine whether pre-existing immunity is present, depending on the different sensitivity requirements.

In certain embodiments, the pre-existing immune response is characterized in that prior to step a), the antibody titer of the anti-mediator antibodies of the serum of the subject is at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200, and wherein the antibody titer is defined as the dilution fold at which the serum of the subject produces a threshold specific binding to the inducer mediator or the target mediator.

In certain embodiments, the inducer vehicle and/or the target vehicle comprise viral particles and the pre-existing immunity can be determined by antibody titer of neutralizing antibodies in the serum of the subject. Neutralizing antibody titers can be analyzed by any suitable method, such as the reporting assays described above. Neutralizing antibody titers can be determined by the dilution factor of serum to produce a maximum neutralization of about 50% of the inducer vehicle and/or target vehicle, optionally present in a given amount. Depending on the different sensitivity requirements, different thresholds may be selected to determine whether pre-existing immunity is present.

In certain embodiments, the pre-existing immune response is characterized by an antibody titer of at least 2, 4, 6, 8, 18, or 32 of neutralizing antibodies in the serum of the subject prior to administration of the inducer vehicle to the subject, and wherein the antibody titer is defined as the dilution fold of the serum of the subject that produces 50% maximum neutralization with the inducer vehicle or target vehicle.

In certain embodiments, the anti-mediator antibody or neutralizing antibody is directed against the capsid of the inducer viral particle of the inducer mediator or against the capsid of the target viral particle of the target mediator or against both.

Method for expressing target gene

In a certain aspect, the disclosure further provides a method of expressing a target gene in a subject, wherein the subject has an existing immune tolerance to the immunogenic target agent. In a certain embodiment, such existing immune tolerance can be induced by the methods provided herein using the immunogenicity inducer vehicles provided herein.

Basically, the method of expressing a target gene in a subject is by an immunogenic target agent comprising a target vector encoding the target gene, wherein the subject is tolerogenic to the immunogenic target agent. The method comprises the following steps: a) Administering an immunogenic target agent to the subject.

Herein, prior to administration of a target vehicle, the subject has been induced to have an existing immune tolerance by exposure to an immunogenicity inducer vehicle provided herein. In certain embodiments, the inducer vehicle comprises an inducer nucleic acid vector encoding at least one immunosuppressant, whereby the at least one immunosuppressant has been expressed in the subject to allow suppression of an immune response directed against the inducer vehicle and cross-reactive with a target viral vehicle.

In a certain aspect, the present disclosure further provides a method of expressing a target gene in a subject by an immunogenic target agent comprising a target vector encoding the target gene. This method is also based on a method of inducing immune tolerance to an immunogenic target agent for gene delivery in a subject as described above. Basically: the method comprises the following two steps:

a) Administering an immunogenic inducer vehicle provided herein to the subject, thereby suppressing an immune response against the inducer vehicle and cross-reactive with the target vehicle such that immune tolerance is induced in the subject against the target vehicle; and

b) Administering the target agent to the subject having such induced immune tolerance, thereby expressing the target gene in the subject.

In certain embodiments, the immunogenic inducer vehicle comprises an inducer nucleic acid vector encoding at least one immunosuppressant, thereby allowing expression of the at least one immunosuppressant in the subject, the at least one immunosuppressant inhibiting an immune response directed against the inducer vehicle and being cross-reactive with the target vehicle such that immune tolerance is induced in the subject against the target vehicle.

Expression of the transgene carried by the inducer/target vector, such as any of the at least one immunosuppressant encoded by the inducer nucleic acid vector and/or any of the at least one target gene encoded by the target nucleic acid vector, can be characterized qualitatively or quantitatively in the subject. For this purpose, general means and methods may be employed which may include qualitative detection of the presence of the transgene in certain organs, body fluids or target cells of the subject and/or quantitative determination of expression levels.

Any convenient method may be used to qualitatively and quantitatively characterize the corresponding polynucleotide of the transgene. For example, polynucleotides can be characterized qualitatively or quantitatively using, for example, PCR (also including quantitative PCR, real-time PCR, etc.), southern blotting, next generation sequencing, microarrays, and the like. If the transgene encodes a gene product such as an RNA product (e.g., mRNA, miRNA, or antisense RNA), expression of the RNA product can be characterized qualitatively or quantitatively by, for example, RT-PCR (including real-time RT-PCR), northern blotting, RNase protection, microarrays, and the like. If the transgenic mRNA product is further translated into a polypeptide product, the polypeptide product can be characterized qualitatively or quantitatively by Western blotting, ELISA, immunohistochemistry, and the like, using a particular antibody directed against each of such polypeptides. Other qualitative and/or quantitative transgene detection techniques and methods may also be employed.

As used herein, the term "expression" or "expression" refers to the transcription of a coding DNA sequence into mRNA and/or the translation of a coding DNA sequence into a peptide or protein. As used herein, the phrase "expression of a transgene" refers to expression of a coding sequence (e.g., a sequence encoding a protein of interest) included in an inducer/target vector. In some embodiments, the expression level of the exogenous nucleic acid is determined based on mRNA levels or protein levels.

In certain embodiments, the expression level of the target gene may be increased, or the duration of expression may be prolonged.

Therapeutic method

In a certain aspect, the present disclosure further provides a method of treating a condition in a subject by an immunogenic target agent comprising a target vector encoding a target gene capable of treating the condition, wherein the subject is tolerogenic to the target agent. The method comprises the following steps: a) Administering to the subject an effective amount of the target agent, thereby expressing the target gene in the subject to treat the condition. Herein, prior to administration of a target vehicle, the subject has been induced to have an existing immune tolerance by exposure to an immunogenicity inducer vehicle provided herein.

In certain embodiments, the inducer vehicle comprises an inducer nucleic acid vector encoding at least one immunosuppressant, whereby the at least one immunosuppressant has been expressed in the subject to allow suppression of an immune response directed against the inducer vehicle and cross-reactive with the target vehicle.

In a certain aspect, the present disclosure further provides a method of treating a condition in a subject by an immunogenic target agent comprising a target vector encoding a target gene capable of treating the condition. The method comprises the following two steps:

a) Administering to the subject an effective amount of an immunogenic inducer vehicle provided herein, thereby inhibiting an immune response against the inducer vehicle and cross-reactive with the target vehicle such that immune tolerance is induced in the subject against the target vehicle; and

b) Administering an effective amount of the target mediator to the subject having such induced immune tolerance, thereby expressing the target gene in the subject to treat the condition.

In certain embodiments, the inducer vehicle comprises an inducer nucleic acid vector encoding at least one immunosuppressant, thereby allowing expression of the at least one immunosuppressant in the subject, the at least one immunosuppressant inhibiting an immune response directed against the inducer vehicle and being cross-reactive with the target vehicle such that immune tolerance is induced in the subject against the target vehicle.

In any of the methods provided in the above aspects of the disclosure, the various embodiments of the inducer vehicle, the at least one immunosuppressant, target vehicle, cross-reactivity, immune response, and/or immune tolerance can be referenced to the various embodiments of the method of inducing immune tolerance to an immunogenic target vehicle for gene delivery in a subject described above. For the sake of brevity, detailed descriptions of various embodiments are omitted herein.

As used herein, the term "condition" refers to any disease or condition in a subject that is susceptible to treatment with a targeting agent that can carry a targeting vector encoding and expressing a target gene. The term "treating" or "treating" a condition as used herein includes alleviating the condition, slowing the onset or rate of progression of the diseased condition, alleviating or ending symptoms associated with the condition, producing complete or partial regression of the condition, curing the condition, or some combination thereof.

In some embodiments, such conditions are characterized by the absence of one or more functional genes or functional proteins. In some embodiments, such conditions are suitable for gene therapy. The target gene carrying the target agent delivered to the subject may be useful for replacing or repairing a deleted or dysfunctional molecular element (e.g., a gene) in the DNA of a living cell of the subject, or alternatively, providing or enhancing the function of the deleted or dysfunctional gene in the cell by introducing and expressing the functional gene in the cell.

In some embodiments, the condition is a "single gene disorder," which is a disorder caused by one or more abnormalities in the genome that affect one or both copies of a single gene. Genomic abnormalities disrupt genes and result in a loss or lack of activity of endogenous proteins encoded by the disrupted genes. Symptoms of monogenic disorders are caused by a lack or deficiency of the activity of endogenous proteins. In some embodiments, the monogenic disorder is autosomal dominant, autosomal recessive, X-linked, Y-linked, or mitochondrial.

Examples of autosomal dominant monogenic disorders include, but are not limited to, brugada Syndrome (Brugada Syndrome), type 1 myotonic dystrophy, type 2 myotonic dystrophy, hereditary multi-infarct dementia, huntington's disease, type 1 neurofibromatosis, type 2 neurofibromatosis, ma Fanzeng Syndrome (Marfan syndrom), familial Hypercholesterolemia (FH), polycystic kidney disease, hereditary spherical erythromatosis, hereditary non-polyposis colorectal cancer, hereditary multiple exotoses, tuberous sclerosis, von willebrand's disease (Von Willebrand disease) and acute intermittent porphyria, delavir Syndrome (Dravet Syndrome), petz-jegstrom Syndrome (Peutz-jegsherherem), chondrogenesis imperfecta, primary combined immunodeficiency, familial Adenomatosis Polyposis (FAP), spinocerebellar ataxia, multiple endocrine tumors.

Examples of autosomal recessive genetic disorders include, but are not limited to, beta-ketothiolase deficiency, biotin enzyme deficiency, hepatolenticular degeneration, spinal muscular atrophy, N-acetylglutamate synthase deficiency, lysosomal acid lipase deficiency, lysine urinary protein intolerance, long chain 3-hydroxyacyl-CoA dehydrogenase deficiency, lelen Syndrome (Laron Syndrome), isovaleric acid Disease, polyornithine-hyperchlorhymia-homoculosidia Syndrome, carboxylated holoenzyme deficiency, hereditary fructose intolerance, glycogen storage Disease type II, type I glutarate, gettman Syndrome (Gitelman Syndrome), gaucher Disease, familial mediterranean fever, gaucher Disease congenital myotonia, citrullinemia I, citrullinemia II, primary carnitine deficiency, arginase deficiency, medium chain acyl-CoA dehydrogenase deficiency, sickle cell Anemia, cystic fibrosis, saxophone's Disease (Tay-Sachs Disease), phenylketonuria, lysosomal acid lipase deficiency, glycogen storage Disease, galactosylemia, niemann-Pick Disease (Niemann-Pick Disease), spinal Muscular Atrophy (SMA), robusts Syndrome (Roberts Syndrome), very long chain acyl-CoA dehydrogenase deficiency, pulmonary cystic fibrosis, gaucher's Disease, vorner Syndrome (Werner Syndrome), fanconi Anemia (Fanconi Anemia), mucopolysaccharidosis (I, IIIA, IIIB, IVA, IVB, VI, VII, IX).

Examples of X-linked monogenic disorders include, but are not limited to, fragile X syndrome, congenital adrenal hypoplasia, duchenne muscular dystrophy and haemophilia a, haemophilia B, fabry Disease, X-linked agaropectinemia, X-linked adrenoleukodystrophy, spinal and bulbar atrophy, ornithine transcarbamylase deficiency and mucopolysaccharidosis II, adrenoleukodystrophy (ALD), chronic granulomatoid Disease.

Other examples of monogenic disorders include Mey-Organd Syndrome (McCune-Albright Syndrome), paroxysmal sleep hemoglobinuria, ADA immunodeficiency, amyotrophic Lateral Sclerosis (ALS), glucose-galactose, muscular dystrophy, azoospermia, ehlers-Danlos Syndrome, retinitis pigmentosa, hemochromatosis, melanoma, retinoblastoma, alzheimer's disease (Alzheimer Disease), amyloidosis, myotonic muscular dystrophy, megaxillary neuropathy, alpha-1 antitrypsin, parkinson's disease, severe combined immunodeficiency (ADA-SCID/X-SCID).

Examples of polygenic disorders include, for example, heart disease, cancer (e.g., leukemia, in particular acute lymphoblastic leukemia), diabetes, schizophrenia, and Alzheimer's disease.

Repeated application

In any of the methods as provided above, the step of administering the target agent to the subject comprises repeatedly administering the target agent to the subject. The time interval between two adjacent administrations may be from 2 weeks to 1 year, and optionally from 1 to 6 months. It is noted, however, that the present disclosure also encompasses embodiments involving repeated administration according to a regular schedule of administration every half-week, every two weeks, or any other regular schedule.

Pharmaceutical compositions and kits

In a certain aspect, the disclosure further provides a kit for inducing immune tolerance of an immunogenic target agent to be delivered in a subject. Such kits can be used to perform the methods of inducing immune tolerance to an immunogenic target agent in a subject as described above.

The kit includes a first pharmaceutical composition comprising a therapeutically effective amount of an inducer vehicle. The inducer vehicle includes at least one inducer nucleic acid vector, each inducer nucleic acid vector encoding and configured to allow expression of at least one immunosuppressant in the subject. Herein, each of the at least one immunosuppressant is characterized in that, upon expression in a subject, the immunosuppressant can suppress an immune response in the subject against an inducer vehicle, and the immune response is configured to be cross-reactive with a target vehicle.

Optionally herein, the inducer vehicle and/or the target vehicle may be a viral-based vehicle comprising viral particles, or may be based on a non-viral vehicle.

In certain embodiments of the kit, the inducer vehicle comprises an inducer viral particle and/or the target vehicle comprises a target viral particle.

Optionally herein, each of the inducer viral particle and the target viral particle may comprise a capsid.

Further optionally, the immune response may include the generation of at least one antibody capable of binding to the capsid of the inducer viral particle.

Further optionally, the at least one antibody may be cross-reactive with the capsid of the target viral particle.

In the kit, both the inducer viral particle and the target viral particle may optionally have the same type of virus. Herein, the type of virus may be selected from the group consisting of: adenoviruses, adeno-associated viruses (AAV), lentiviruses, retroviruses, and oncolytic viruses. In certain embodiments, the type of virus is an adeno-associated virus (AAV). Furthermore, both the inducer viral particle and the target viral particle may be based on AAV having serotype AAV 8.

In certain embodiments of the kit, the inducer viral particle and the target viral particle are of the same serotype or of different serotypes.

In certain embodiments of the kit, each of the inducer viral particle and the target viral particle comprises the same capsid protein.

In any of the embodiments of the kits described above, the at least one immunosuppressant can act by downregulating an immunostimulatory pathway and/or by upregulating an immunosuppressive pathway.

According to some embodiments, one or more of the at least one immunosuppressant reduces expression or activity of one or more targets in the immunostimulatory pathway. Optionally, each of the one or more targets is selected from the group consisting of: CD27, CD70, CD28, CD80 (B7-1), CD86 (B7-2), CD40L (CD 154), CD122, CD137L, OX (CD 134), OX40L (CD 252), GITR, ICOS (CD 278) and ICOSLG (CD 275).

Further optionally, the one or more targets are in at least one of a B7/CD28 costimulatory signaling pathway or a CD40/CD40L costimulatory signaling pathway.

Further optionally, each of the one or more targets is selected from the group consisting of: CD28, CD80 (B7-1), CD86 (B7-2), CD40 or CD40L (CD 154).

According to some other embodiments, one or more of the at least one immunosuppressant increases expression or activity of one or more targets in the immunosuppression pathway. Optionally, each of the one or more targets is selected from the group consisting of: a2AR, B7-H3 (CD 276), B7-H4 (VTCN 1), BTLA (CD 272), CTLA-4 (CD 152), IDO1, IDO2, TDO, KIR, LAG3, NOX2, PD-1, PD-L2, TIM-3, VISTA, SIGLEC7 (CD 328), TIGIT, PVR (CD 155) and SIGLEC9 (CD 329).

According to still other embodiments, the at least one immunosuppressant modulates the expression or activity of at least one of TLR2, TLR9, myD88, IFN-1, IRF-7, NF- κ B, mTOR, CD3, CD4, CD278, CD19, CD20, CD79, IL-4, IL-2R, IL-5, IL-6R, TNF- α or LFA-1.

In certain embodiments, the at least one immunosuppressant down-regulates the B7-CD28 immunostimulatory pathway. Herein, optionally, the at least one immunosuppressant inhibits expression and/or function of CD28, CD80 (B7-1), CD86 (B7-2). In certain embodiments, the at least one immunosuppressant disrupts or blocks B7-CD28 interactions (e.g., binding). In certain embodiments, the at least one immunosuppressant competes with CD28 for binding to B7, or competes with B7 for binding to CD 28.

In certain embodiments, the at least one immunosuppressant comprises a CTLA-4 derivative. Optionally, the at least one immunosuppressant comprises an extracellular domain of CTLA-4, a B7 binding fragment of CTLA-4, or a mutant thereof. Various embodiments of suitable CTLA-4 derivatives may be referred to elsewhere, for example, in the methods described in other aspects of the disclosure.

In certain embodiments, the at least one immunosuppressant down-regulates the CD40/CD40L immunostimulatory pathway. In certain embodiments, the at least one immunosuppressant inhibits expression and/or function of CD40 or CD 40L. In certain embodiments, the at least one immunosuppressant disrupts or blocks CD40 and CD40L interactions. In certain embodiments, the at least one immunosuppressant competes with CD40L for binding to CD40, or competes with CD40 for binding to CD 40L. In certain embodiments, the at least one immunosuppressant comprises a CD40L binding ligand or a CD40L binding fragment of an anti-CD 40L antibody.

In certain embodiments, the at least one immunosuppressant comprises a CD40 derivative. Optionally, the at least one immunosuppressant comprises an extracellular domain of CD40, a CD 40L-binding fragment of CD40, or a mutant thereof which retains CD40L binding function or affinity. Various embodiments of suitable CD40 derivatives may be referred to elsewhere, for example, in the methods described in other aspects of the disclosure.

In certain embodiments of the kit, the at least one immunosuppressant comprises at least one immunosuppressive protein.

In certain embodiments, the at least one immunosuppressive protein comprises one of the following, or a combination comprising two immunosuppressive proteins:

Further optionally, each immunosuppressive protein of the at least one immunosuppressive protein further comprises a half-life extending peptide moiety. In certain embodiments, the half-life extending peptide moiety comprises an Fc moiety, such as an IgG Fc. Other embodiments of half-life extending peptide moieties may include albumin family member proteins (e.g., HSA, etc.), follicle Stimulating Hormone (FSH), fibronectin, fibroblast Activation Protein (FAP), coagulation factors (e.g., von willebrand factor, factor V, etc.), or functional (i.e., capable of extending half-life) fragments, and also include serum albumin binding polypeptides. For more details, reference may be made to other parts of other aspects of the disclosure, for example, the methods as described above.

In certain embodiments of the kit wherein each of the inducer vehicle and the target vehicle comprises a viral particle, the kit may optionally further comprise a packaging cell line configured to allow production of the inducer vehicle comprising each of the at least one inducer nucleic acid vector.

Optionally herein, the viral particles are derived from an adeno-associated virus (AAV), and optionally the viral particles are derived from a packaging cell line, optionally a HEK293 cell line or variant thereof. Many variants of HEK293 cell lines have been reported, for example, HEK293 (e.g., ATCC crl-1573); HEK293T (e.g., ATCC CRL-11268; ACS-4500, CRL-11268G-1; ATCC CRL-3216); HEK293 STF (ATCC CRL-3249); HEK-293.2sus (ATCC CRL-1573.3); AAVpro 293T (TaKaRa catalog number 632273); HEKExpress (excel gene); 293AAV Cell lines (Cell Biolabs, catalog number AAV-100), and the like. These can be used for AAV production.

In any of the embodiments of the kits described above, the kit may further comprise a first instruction for administering the first pharmaceutical composition to the subject. Herein, the first instructions may include information specifying the dose and/or the route/mode of delivery of the first pharmaceutical composition to be administered to the subject.

In any of the embodiments of the kits described above, the kit may further comprise a first detection kit for determining whether to induce the immune tolerance to the target agent in the subject. In certain embodiments, wherein the immune response to the inducer vehicle and the immune response being cross-reactive with the target vehicle comprises generating at least one anti-vehicle antibody or at least one neutralizing antibody capable of binding to the inducer vehicle and cross-reacting with the target vehicle. Thus, the first detection kit may further comprise at least one first reagent configured to allow determination of the titer of one or more of the anti-mediator antibodies and/or one or more of the neutralizing antibodies in the serum of the subject. Herein, one or more of the at least one antibody may optionally comprise an antibody capable of neutralizing the inducer vehicle and/or the target vehicle. Information about whether a purported immune tolerance was induced in the subject following administration of the first pharmaceutical composition to the subject is provided by a first detection kit. If it is shown that an acceptable level of immune tolerance is induced in the subject, the subject may then further undergo subsequent administration of the immunogenic target agent, resulting in increased efficiency and efficacy of delivery of the target agent in the subject.

Optionally herein, the kit may further comprise a second detection kit comprising at least one second reagent configured to allow detection of expression of each of the at least one immunosuppressant in the subject.

In any of the embodiments of the kits described above, the kit may further comprise a second pharmaceutical composition comprising a therapeutically effective amount of the target agent, and may optionally further comprise a second instruction for administering the second pharmaceutical composition to the subject. Herein, the second instructions may include information specifying the dose and/or route/mode of delivery of the second pharmaceutical composition to be administered to the subject. Further optionally, the second instructions may further comprise a first sub-instruction for administering the second pharmaceutical composition to the subject after administration of the first pharmaceutical composition.

Optionally, the second instructions may further comprise a second sub-instruction for repeated administration of the second pharmaceutical composition. Herein, the second sub-instructions may specify information such as the repeated dose, the delivery pattern of each subsequent administration, and the frequency of repeated administration, i.e. the time interval between each two adjacent administrations of the second pharmaceutical composition.

In any of the above embodiments, the targeting agent included in the second pharmaceutical composition may include a targeting vector encoding a target gene and configured to allow expression of the target gene in a subject. In other words, the second pharmaceutical composition may allow delivery and expression of the target gene in the subject. Optionally herein, the kit may further comprise a third detection kit comprising at least one third reagent configured to allow detection of expression of the target gene in the subject. The at least one third reagent may be used herein for any suitable gene expression analysis, which may include Northern blot analysis, quantitative PCR analysis, western blot analysis, microarray analysis (e.g., illumina or Affymetrix), next generation analysis, protein chip analysis, or the like, without limitation herein.

As an alternative to the above-described embodiments in which the inducer and/or target agent comprises a viral agent, in certain embodiments of the kit the inducer and/or target agent may comprise a non-viral nanocarrier such as a synthetic nanocarrier. For more details, reference may be made to methods described elsewhere in this disclosure.

In a certain aspect, the disclosure further provides a kit for expressing a target gene in a subject.

A kit comprises a pharmaceutical composition comprising an immunogenic target agent, and the target agent comprises a target vector encoding the target gene and configured to allow expression of the target gene in the subject.

The kit further includes instructions for administering a pharmaceutical composition to a subject, the instructions including a method for inducing immune tolerance to the target agent prior to administering the pharmaceutical composition to the subject. The method comprises the following steps: a) Administering an immunogenic inducer vehicle to the subject, wherein the inducer vehicle comprises an inducer nucleic acid vector encoding at least one immunosuppressant, thereby allowing expression of the at least one immunosuppressant in the subject, the at least one immunosuppressant inhibiting an immune response in the subject against the inducer vehicle, wherein the immune response is cross-reactive with the target vehicle. Herein, the delivery instructions may contain information specifying the dosage and/or route/mode of delivery of the pharmaceutical composition.

Optionally, the kit may further comprise a first detection kit for determining whether an immune response in an immunocompromised subject is induced in the subject.

In embodiments where the immune response includes the production of at least one antibody capable of binding to and cross-reacting with the inducer vehicle, the first detection kit may include at least one first reagent configured to allow determination of the titer of one or more of the at least one antibody in the serum of the subject. Herein, one or more of the at least one antibody may comprise an antibody capable of neutralizing an inducer vehicle and/or a target vehicle.

In certain embodiments, the kit may further comprise a third detection kit comprising at least one third reagent configured to allow detection of expression of the target gene in the subject.

In any of the embodiments of the kit described above in this aspect, the instructions may further comprise sub-instructions for repeated administration of the pharmaceutical composition. Herein, the sub-instructions may specify information such as the repeated dose, the mode of delivery of each subsequent administration, and the frequency of repeated administration, i.e., the time interval between each two adjacent administrations of the pharmaceutical composition.

In a certain aspect, the disclosure further provides a kit for expressing a target gene in a subject by an immunogenic target mediator, the immunogenic target mediator encoding the target gene.

The kit includes a first pharmaceutical composition for inducing immune tolerance to the target agent in the subject and a second pharmaceutical composition comprising the target agent. The first pharmaceutical composition comprises an inducer vehicle comprising at least one inducer nucleic acid vector, each inducer nucleic acid vector encoding at least one immunosuppressant and being configured to allow expression of the at least one immunosuppressant in the subject, and each of the at least one immunosuppressant being configured to inhibit an immune response in the subject against the inducer vehicle when expressed in the subject, and the immune response being cross-reactive with the target vehicle. The targeting agent comprises a targeting vector encoding a target gene.

Optionally, the kit further comprises a first detection kit for determining whether the immune tolerance is induced in the subject after administration of the first pharmaceutical composition to the subject.

In embodiments where the immune response includes the production of at least one antibody capable of binding to and cross-reacting with the inducer vehicle, the first detection kit may include at least one first reagent configured to allow determination of the titer of one or more of the at least one antibody in the serum of the subject.

The kit may optionally further comprise a second detection kit comprising at least one second reagent configured to allow detection of expression of each of the at least one immunosuppressant in the subject.

The kit may optionally further comprise a third detection kit comprising at least one third reagent configured to allow detection of expression of the target gene in the subject.

In any of the embodiments as described above, the kit may further comprise first instructions for administering the first pharmaceutical composition; and a second instruction for administering the second pharmaceutical composition. Herein, the first and second instructions may include information specifying the delivered dose and/or route/pattern of delivery of the first and second pharmaceutical compositions, respectively.

Optionally, the second instructions further comprise a first sub-instruction for administering the second pharmaceutical composition to the subject after administration of the first pharmaceutical composition.

Further optionally, the second sub-instructions further comprise a second sub-instruction for repeated administration of the second pharmaceutical composition. Herein, the second sub-instructions may specify information such as the repeated dose, the delivery pattern of each subsequent administration, and the frequency of repeated administration, i.e. the time interval between each two adjacent administrations of the second pharmaceutical composition.

In any embodiment of the kit for any of the various aspects described above, the kit may optionally further comprise a third pharmaceutical composition comprising at least one immunosuppressive agent. Each of the at least one immunosuppressive agent is configured to be capable of inhibiting or enhancing the activity of a target in an immunostimulatory pathway. Optionally herein, each of the at least one immunosuppressive agent comprises a compound, a nucleic acid, a polypeptide, or a combination thereof. In certain embodiments of the kit, each of the at least one immunosuppressant and the at least one immunosuppressant agent is configured to have a different target to achieve a synergistic effect, thereby achieving improved immunosuppression.

In various embodiments of kits for the various aspects described above, the pharmaceutical composition, the at least one first reagent, the at least second reagent, and the at least third reagent will be described in more detail in the following sections.

As used herein and in the broadest sense, the term "pharmaceutical composition" is meant to include a therapeutically effective amount of a pharmaceutically active ingredient and a formulation, optionally together with pharmaceutically acceptable excipients and/or carriers, and the like. As used herein, the term "pharmaceutically acceptable" means suitable for normal pharmaceutical use, i.e., without causing serious side effects such as adverse events in a subject. As used herein, the term "therapeutically effective amount" or "effective amount" means a dosage sufficient to render a patient therapeutically effective as compared to no treatment. As used herein, an "effective amount" refers to an amount that produces a desired effect, such as being capable of treating or ameliorating a disease or condition or otherwise producing a desired therapeutic effect (e.g., inducing immune tolerance).

The pharmaceutically active ingredient typically requires one or more pharmaceutically acceptable carriers or excipients in order to form a suitable form (i.e., pharmaceutical composition) and dosage form for effective administration to a subject. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not negate the biological activity and properties of the administered pharmaceutically active agent. Herein, the term "pharmaceutically acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a pharmaceutically active ingredient.

Suitable pharmaceutical compositions herein include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the compositions must be sterile and stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The pharmaceutically acceptable carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like) and suitable mixtures thereof. For example, for intravenous administration, suitable carriers may include physiological saline, bacteriostatic water, or Phosphate Buffered Saline (PBS). Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. The prevention of the action of microorganisms can be achieved by various antibacterial agents and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like). In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride in the composition. Prolonged absorption of the internal composition may be achieved by including agents in the composition that delay absorption, such as aluminum monostearate and gelatin.

Sterile solutions may be prepared by incorporating the active compound in the required amount in the appropriate solvent with one or a combination of the components enumerated above, followed by sterile filtration, as required. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In one embodiment, the active compounds are prepared with carriers that will protect the compounds from rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid may be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

Pharmaceutical compositions may include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be produced from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids.

In some embodiments, the pharmaceutical compositions provided herein comprise a viral particle or virion (e.g., a rAAV or adenovirus vector) as a pharmaceutically active ingredient. Viral particles typically include a nucleic acid vector and a viral capsid. Herein, the nucleic acid vector may encode at least one immunosuppressant (for an inducer vector) or target gene (for a target vector), the viral capsid comprises one or more viral capsid proteins, and the nucleic acid vector is packaged or encapsulated into a viral capsid, thereby forming a viral particle.

Viral particles (e.g., recombinant AAV) can be incorporated into a pharmaceutical composition for administration to a mammalian patient, particularly a human. The virions can be formulated in a non-toxic inert pharmaceutically acceptable aqueous carrier, optionally at a pH in the range of 3 to 8, such as in the range of 6 to 8. Such a sterile composition will include a vector or virosome containing nucleic acid encoding a therapeutic molecule that is dissolved in an aqueous buffer having an acceptable pH after reconstitution.

In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of virosomes in admixture with pharmaceutically acceptable carriers and/or excipients, such as saline, phosphate buffered saline, phosphates and amino acids, polymers, polyols, sugars, buffers, preservatives and other proteins. Exemplary amino acids, polymers, and sugars are octyl phenoxy polyethoxy ethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, ringer's and hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycols. In certain embodiments, this formulation is stable for at least six months at 4 ℃.

In some cases, unit doses of the pharmaceutical compositions of the present disclosure can be measured as pfu (plaque forming units). In some cases, the pfu of a unit dose of a pharmaceutical composition of the present disclosure comprising an inducer vehicle may be about 1 x 10 ⁸ Up to about 5X 10 ¹² pfu. In some cases, pfu of a unit dose of a pharmaceutical composition of the present disclosure comprising a target agent may be at a dose higher than the dose of the inducer agent. In some cases, a unit dose pfu of a pharmaceutical composition of the present disclosure comprising a targeting agent may be 1 x 10 ⁸ Up to about 1X 10 ¹⁴ pfu。

In some embodiments, the vector comprises a viral vector. In some cases, the viral vectors of the present disclosure may be measured as vector genomes.

In some cases, the pharmaceutical compositions of the present disclosure comprising an inducer vehicle have a unit dose of about 1 x 10 ⁸ Up to about 5X 10 ¹² A single vector genome or more. In some embodiments, the present disclosure comprising a targeting agentThe unit dose of the pharmaceutical composition of (2) is 1×10 ⁸ Up to about 1X 10 ¹⁴ And a vector genome.

In some aspects, the pharmaceutical composition comprising the inducer vehicle comprises about 1 x 10 ⁸ Up to about 1X 10 ¹² And (3) recombinant viruses. In some aspects, a pharmaceutical composition comprising a target agent comprises 1 x 10 ⁸ Up to about 1X 10 ¹⁴ And (3) recombinant viruses.

In certain embodiments, a non-viral based delivery system is used in one or both of the inducer and target vehicles in the methods and kits provided in the present disclosure.

The compositions provided herein may comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphates, carbonates, acetates, or citrates) and pH adjusting agents (e.g., hydrochloric acid, sodium or potassium hydroxide, citrates or acetates, amino acids, and salts thereof), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), solution and/or freeze/lyophilization stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjusting agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsiloxane), preservatives (e.g., thimerosal (thimerosal), 2-phenoxyethanol, EDTA), polymer stabilizers, and viscosity adjusting agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethyl cellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol).

The composition according to the invention may comprise pharmaceutically acceptable excipients. The compositions may be prepared using conventional pharmaceutical manufacturing and compounding techniques to obtain useful dosage forms. Techniques suitable for practicing the present invention can be found in the industrial mixing handbook: science and practice (Handbook of Industrial Mixing: science and Practice), edited by Edward L.Paul, victor A.Atiemo-Obeng and Suzanne M.Kresta, 2004 John Wiley & Sons, inc.); pharmaceutical science: formulation science (pharmaceuticals: the Science of Dosage Form Design), 2 nd edition, edited by M.E. Auten, 2001, qiugillivenston corporation (Churchill Livingstone). In one embodiment, the composition is present with a preservative in a sterile saline solution for injection.

It should be understood that the compositions of the present invention may be prepared in any suitable manner and that the present invention is in no way limited to compositions that may be produced using the methods described herein. The selection of an appropriate manufacturing method may require attention to the characteristics of the particular portion associated.

In some embodiments, the composition is manufactured under aseptic conditions or terminally sterilized. This ensures that the resulting composition is sterile and infection-free, thereby improving safety when compared to non-sterile compositions. This provides a valuable safety measure, especially when the subject receiving the composition is immunodeficient, suffering from an infection and/or susceptible to an infection. In some embodiments, depending on the formulation strategy, the composition may be lyophilized and stored in suspension or lyophilized powder form for extended periods of time without losing activity.

The methods of use, i.e., administration, of these aforementioned synthetic nanocarriers to carry target cargo to a subject can take a variety of routes, which can be systemic or in a localized treatment area. Optionally, the subject may be administered via parenteral, oral, enteral, intraoral, nasal, topical, rectal, vaginal, transmucosal, epidermal, transdermal, dermal, ocular, pulmonary, cardiac, subcutaneous, intraparenchymal, intraventricular, or intrathecal routes of administration. In certain embodiments, the exogenous nucleic acid is delivered to the subject orally, intravenously, intraperitoneally, intramuscularly, subcutaneously, intrathecally, intraventricularly (ICV), or intrarectally. The route of administration may also include administration by inhalation or pulmonary aerosol. Techniques for preparing aerosol delivery systems are well known to those skilled in the art.

Method for characterizing certain substances

In any of the embodiments of the methods, kits, or pharmaceutical kits described above, certain substances may be characterized qualitatively or quantitatively after administration to a subject to determine one or more of the following:

a) The level of pre-existing host immune response to the target agent can be determined by quantitatively characterizing the antibody, particularly the antibody titer of neutralizing antibodies, to the target agent (e.g., anti-capsid antibody as in the case of AAV vectors) in a serum sample of the subject;

b) The effectiveness of delivery of an inducer vehicle to a subject can be determined by characterizing the expression of the at least one immunosuppressant (e.g., CTLA4-Ig and/or CD 40-Ig) encoded by the inducer nucleic acid vector;

c) Expression of the at least one immunosuppressant has an immunosuppressive effect (i.e., a level of immune tolerance induced) on a host immune response that is cross-reactive to the inducer and/or to the target vehicle, as determined by quantitatively characterizing antibody, particularly neutralizing antibody, titers of antibodies to the inducer/target vehicle (as in the case of AAV vectors) in a serum sample of the subject; and/or

d) The effectiveness of delivery of the target agent to the subject can be determined by characterizing the expression of the at least one target gene encoded by the target nucleic acid vector.

An example is provided below as one specific embodiment of the methods and kits provided in the present disclosure. Note that the examples are for illustration purposes only and do not limit the scope of the present disclosure.

Example 1

In this example, when mice were treated by single administration of AAV8 vectors carrying CTLA4-Ig alone or CTLA4-Ig and CD40-Ig, in vivo studies of GFP gene expression were performed by repeated AAV8 delivery. In this process, humoral immune responses and immune tolerance of AAV8 were also estimated.

Materials and methods

AAV vector preparation

The transgenic plasmid was constructed by ligating the extracellular domain of mouse CTLA4 or CD40 to the Fc portion of mouse IgG (defined as CTLA4-Ig or CD40-Ig, wherein the sequences are shown in SEQ ID NOs: 8 and 9, respectively) as previously reported (Ye X et al 2005). The AAV8 vector genome contains the AAV2 terminal repeat, the CAG promoter (CMV enhancer/chicken actin promoter/human globin intron), the SV40 splice signal, the transgene coding sequence CTLA4-Ig or CD40-Ig, and the polyadenylation signal. AAV8 vector production, purification and titration were performed as previously disclosed (Wang B et al, 2000).

Animal and treatment

C57BL/6J 6-8 week old WT male mice were purchased from Collection Cuff Biotechnology (JiCui Biology Company) (Nanj, china). All mice were housed under standard conditions and all procedures were performed following guidelines of the institutional animal care and use committee (University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee) of the church mountain school of university of north carolina.

In the first administration experiment, mice (n=5 per group) were injected via the tail vein with two doses of 4×10 ¹² vg/kg (high dose, HD) and 4X 10 ¹¹ vg/kg (medium dose, MD). The virus combinations were divided into four groups: AAV8-CTLA4-Ig, AAV8-CD40-Ig, AAV8-CTLA4-Ig+AAV8-CD40-Ig, AAV8-GFP, wherein AAV8-GFP was used as a control group. In the second and third administration experiments at week 8 (defined as "8 w") and week 15 (defined as "15 w"), respectively, all groups of mice were administered 6×10 by intravenous injection ¹¹ vg/kg AAV8-GFP. Mice were bled weekly to collect serum or plasma, and eventually sacrificed after 25 weeks.

To verify whether AAV8-CTLA4-Ig or AAV8-CD40-Ig immunomodulatory treatment could effectively inhibit antibody production to AAV vectors of other serotypes, mice were injected intravenously at week 20 (defined as "20 w") with 4X 10 ¹² vg/kg AAV843 (AAV 843-GLUC) expressing Gaussian luciferase. Mice were bled weekly to obtain plasma or serum. And monitoring GLUC expression in plasma according to the published protocol of Bakhos et al (Bakhos a et al 2009).

To elucidate the dynamic changes in exogenous transgene expression by multiple injections of AAV vectors, mice were preimmunized with AAV-CTLA4-Ig or AAV-CTLA4+ AAV-CD40-Ig and AAV-GFP was used as a control, with high and medium doses of 4X 10, respectively ¹² vg/kg (HD) and 4X 10 ¹¹ vg/kg (MD). After 4 weeks, mice were given two injections 6×10 intravenously every 3 weeks ¹¹ vg/kg AAV-GLUC. Mouse plasma was collected to observe GLUC expression.

Western blot

HEK293 cells were transduced with the constructed CTLA4-Ig or CD40-Ig plasmids, and HUH-7 cells were transfected with AAV8 containing the CTLA4-Ig or CD40-Ig expression cassette. The protein from the cells was extracted using RIPA lysis buffer (50 mM Tris-HCl, pH7.5, 150mM NaCl,0.1% NP-40,0.5% sodium deoxycholate, 1% sodium dodecyl sulfate) and protease inhibitor cocktail (Bimake), while the protein from the cell culture media supplemented with protease inhibitors was extracted. Protein concentration was detected by BCA kit (Thermo). Protein samples were separated using a 10% gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with HRP conjugated secondary antibody (1:5000 dilution, ai Bokang company (Abcam)) for two hours at room temperature in tween-added Triple Buffered Saline (TBST) with 5% skim milk powder, and finally protein bands were detected with ECL western blot substrate (tenat).

ELISA

The CTLA4-Ig and CD40-Ig levels in cell culture media, cell lysates and mouse serum were quantified using a mouse CTLA4 ELISA kit (R & D Systems, minneapolis, MN) or a mouse CD40 ELISA kit (SAB, college Park, MD), respectively. The total IgG levels in the mouse serum were measured using a mouse IgG ELISA kit (mlbi, shanghai, china), shanghai, china. All procedures were performed according to the manufacturer's instructions. Finally, the OD of the sample was read at 450 nm. Cytokines including TNF- α, IL-4 and IL-10 in mouse plasma were assayed using Luminex X-200 (R & D company of Minneapolis, minnesota), and data analysis was determined using MILLIPLEX according to the manufacturer's instructions.

AAV neutralization assay

To explore neutralizing antibodies against AAV vectors, mice were bled weekly to analyze serum samples. In short, willSerum samples were incubated at 56℃for 30 minutes to inactivate complement. Serum samples were diluted in round bottom 96 well plates starting at 1:2, mixed with AAV vector expressing luciferase and placed at 37 ℃ in 5% CO ₂ The incubator was maintained for one hour. Next, a volume of carrier serum mix plate was transferred to HUH-7 cell plates. After one hour, 20% FBS complete medium was carefully added and left to stand at 37℃with 5% CO ₂ The incubator was left overnight. After 72 hours, the activity of the luciferase was measured. Herein, a neutralizing antibody titer below 1:8 (8-fold dilution of serum causes inhibition of vector transduction beyond 50%) indicates negative NAb levels.

Binding ELISA

To explore IgG against AAV8 capsids, bound IgG in mouse serum was determined according to published procedures (Murrey DA et al, 2014). Briefly, will contain 1X 10 ¹⁰ The individual particles/mL of empty AAV8 capsid carbonate buffer were pre-coated on 96-well plates overnight at 4 ℃. Samples with AAV were used as positive antigens and carbonate buffer was used as negative antigen only. After blocking with 5% milk powder in Tween-20 in PBS (PBST), serum samples were diluted at 1:50, added to the plates and incubated for one hour at room temperature. Next, the plates were washed with PBST and then incubated with HRP conjugated anti-mouse IgG (Sigma-Aldrich, st Louis, MO) for one hour at room temperature. Plates were washed with PBST added substrate TMB (sigma-aldrich company of st lewis, missouri) for 10 minutes at room temperature. Finally, the sample was terminated with 1N sulfuric acid and the OD was read at 450 nm. anti-AAV 8-IgG titers were calculated as follows: (OD of positive antigen-OD of negative antigen)/OD of negative antigen, wherein a result below 2 is considered a negative anti-AAV-IgG level (in other words, threshold specific binding is set to 2).

Immunofluorescence

To examine the transfection efficiency of AAV8 vectors in vivo or transgene expression following repeated administration of AAV8, the fluorescence intensity of Green Fluorescent Protein (GFP) in tissues was observed. At the end of the study, all groups of mice died. Mouse livers, muscles were collected, fixed in 4% paraformaldehyde, dehydrated in 30% sucrose, and 8- μm frozen or paraffin sections were prepared, which were observed under confocal laser scanning microscopy (Nikon inc., melville, NY, USA) at 20x magnification.

Flow cytometry

To observe immune T-cell and B-cell status, single cell suspensions obtained from mice peripheral blood and spleen were stained with surface markers: anti-CD 3 (APC-Cy 7), anti-CD 4 (FITC), anti-CD 19 (PE), anti-B220 (APC), anti-GL 7 (BV 421), anti-CD 25 (APC) and FoxP3 intracellular staining (PE). All antibodies were purchased from BD Biosciences (BD Biosciences) and the procedure was performed according to the manufacturer's instructions. The samples were observed by CytoFLEX flow cytometry (Beckman Coulter inc., brea, CA). The data were analyzed using cyt expert 2.0 software (beckmann coulter, braziia, california) OR FlowJo software (Tree Star, ashland, OR), ashland, oregon.

Statistical analysis

Data are shown as mean ± SEM values. Statistical analysis was performed using GraphPad Prism v7 (GraphPad software company of lajola, california (GraphPad Software inc., la Jolla, CA)). One-way analysis of variance (ANOVA) and unpaired stink t test (Student t test) were performed for multiple sets of comparisons. P values less than 0.05 (x) or 0.01 (x) are considered statistically significant.

Results

AAV8 delivery CTLA4-Ig or CD40-Ig is stably expressed in vivo

In this example, pAAV-CTLA4-Ig and pAAV-CD40-Ig (FIG. 1A) were constructed and transfected into HEK293 cells. Cells and media were assayed by western blot and ELISA 72 hours after transfection. The results show that CTLA4-Ig and CD40-Ig are significantly up-regulated in transfected cells compared to untransfected cells, and that the two recombinant proteins are secreted in the medium (fig. 1B). When HUH-7 cells were infected with AAV8-CTLA4-Ig or AAV8-CD40-Ig, both proteins were also strongly expressed in the cells and efficiently secreted into the culture medium (FIG. 1C).

Further, two doses (HD and MD) of AAV8-CTLA4-Ig, AAV8-CD40-Ig, or AAV8-CTLA4-Ig+AAV8-CD40-Ig were injected intravenously into mice. ELISA assays showed increased CTLA4-Ig expression in the HD group and peaked at week 8 (PFAW) after the first administration, with concentrations of 600-800 μg/mL. Thereafter, the serum concentration gradually decreased and was maintained at 350-600. Mu.g/mL (FIG. 1D). CD40-Ig expression in the HD group gradually increased and reached a peak at PFAW 12, with a concentration of 10-15 μg/mL, followed by a decrease in concentration and maintenance at 2.5-10 μg/mL (FIG. 1E). Interestingly, CTLA4-Ig concentrations in serum were about 60-fold higher than CD40-Ig. Since the corresponding dose is halved, CTLA4-Ig and CD40-Ig in the AAV8-CTLA4-Ig+AAV8-CD40-Ig group are less than CTLA4-Ig and CD40-Ig in the AAV8-CTLA4-Ig or AAV8-CD40-Ig group. In addition, the in vivo transfection efficiency of AAV8 vectors by different injection routes is shown in fig. 1F. These results demonstrate that CTLA4-Ig and CD40-Ig are stably and efficiently expressed by AAV8 delivery in vitro and in vivo.

Continuous expression of CTLA4-Ig and CD40-Ig inhibits humoral immunity of AAV8 after multiple systemic administration

NAb levels after administration of AAV8-CTLA4-Ig, AAV8-CD40-Ig and AAV8-CTLA4-Ig+AAV8-CD40-Ig were studied at high and medium doses. Further, a strategy for estimating inhibition of antibodies after multiple systemic administrations of AAV8-GFP vector exposure was established (fig. 2A). Serum NAb titers were measured weekly after primary AAV8 vector injection. The results show that mice in both doses of AAV8-CTLA4-Ig and AAV8-CTLA4-Ig + AAV8-CD40-Ig groups had lower NAb compared to the control group treated with AAV8-GFP (fig. 2B, × p < 0.01). Although NAb titers were between 1:10 and 1:25 at PFAW 1 in the AAV8-CTLA4-Ig and AAV8-CTLA4-ig+aav8-CD40-Ig groups, later dropped to less than 1:5 (NAb negative) (fig. 2B). At PFAW 8, AAV8-GFP is injected into mice expressing immunosuppressants including CTLA4-Ig, CD40-Ig, CTLA4-Ig+CD40-Ig and GFP groups (controls). The results show that NAb titers in the AAV8-CTLA4-Ig and AAV8-CTLA4-Ig + AAV8-CD40-Ig groups were significantly enhanced at PFAW 9 and then decreased compared to the initial administration (fig. 2B). However, it was noted that after a second exposure to AAV8-GFP vector at PFAW 16, the NAb titers in AAV8-CTLA4-Ig and AAV8-CTLA4-Ig+AAV8-CD40-Ig groups increased slightly, followed by a recovery to negative levels (FIG. 2B). Furthermore, during these multiple administrations of AAV8 vector, NAb titers in the AAV8-CTLA4-Ig group were not different from that in the AAV8-CTLA4-ig+aav8-CD40-Ig group, but there was dose-dependent inhibition of NAb production in HD and MD groups. In the AAV8-CD40-Ig group, NAb levels were also lower than in the control group, but there was no difference between the two groups (p=0.089). In the absence of immunosuppressant, mice of the control group treated with AAV8-GFP alone were positive NAb (> 1:32). The results show that CTLA4-Ig or CTLA4-Ig and CD40-Ig long-term expression is effective in inhibiting the dramatic increase in NAb and remains at negative levels after single and multiple AAV8 injections.

Further, the overall levels of anti-AAV 8 IgG were analyzed from week 5 of primary AAV8 vector administration. Mice in the AAV8-CTLA4-Ig and AAV8-CTLA4-Ig + AAV8-CD40-Ig groups had lower levels of anti-AAV 8 IgG compared to the control group (p < 0.01), while in the AAV8-CD40-Ig group, the anti-AAV 8 IgG levels were slightly lower but not significantly different (fig. 2c, p=0.057). These data are consistent with NAb assays. Although all groups of mice had an increase in anti-AAV 8 IgG during the first week of exposure to each injection of AAV8 vector, thereafter the AAV8-CTLA4-Ig and AAV8-CTLA4-Ig+AAV8-CD40-Ig groups had a decrease in anti-AAV 8 IgG and were maintained at levels of less than 1:100. anti-AAV 8 IgG has high levels (> 1:1600) in the control group (fig. 2C).

There is no detailed description of the relationship between AAV NAb and anti-AAV capsid IgG (Kruzik a et al, 2019), and therefore AAV8 NAb titers were analyzed for correlation with anti-AAV 8-IgG levels (fig. 2D), and were found to be highly correlated with each other (n=5, r ² ＝0.8451，p<0.0001 This means that both NAb and anti-AAV capsid IgG are characteristic of humoral immunity of the AAV vector.

Taken together, these results demonstrate that delivery of AAV8-CTLA4-Ig or AAV8-CTLA4-ig+aav8-CD40-Ig allows continuous expression of CTLA4-Ig or CTLA4-Ig and CD40-Ig in vivo, which strongly inhibits antibodies to AAV8 and gradually induces immune tolerance to AAV8 in mice.

Continuous expression of CTLA4-Ig or CD40-Ig does not impair the overall humoral immunity

To investigate the effect of long-term CTLA4-Ig or CD40-Ig expression on humoral immunity under single or multiple AAV vector administrations, total mouse IgG and inflammatory cytokines including TNF- α, IL-4 and IL-10 from peripheral blood were analyzed at the first and second injections of AAV8-GFP vector. Total mouse IgG was assayed weekly for 11 weeks (fig. 3A), without significant differences between immunosuppressant treated and control groups (p > 0.05). Plasma was collected on days 4 and 7 of secondary AAV8-GFP administration to test for inflammatory cytokines (fig. 3B, 3C and 3D). The results show no significant differences (p > 0.05) between TNF- α, IL-4 and IL-10 levels in the immunosuppressant group and the control group. In conclusion, continuous exogenous expression of CTLA4-Ig and CD40-Ig via AAV8 delivery does not impair normal humoral immunity.

Immunosuppression of CTLA4-Ig and CD40-Ig improves transgene expression upon repeated administration of AAV8 vectors

At PFAW 25, mice were sacrificed to obtain liver and muscle tissue for observation of GFP expression. In the liver, GFP was expressed in the AAV8-CTLA4-Ig+AAV8-CD40-Ig and AAV8-CTLA4-Ig groups, but not in the AAV8-CD40-Ig groups (FIGS. 4A and 4B). In both liver and muscle tissue, AAV8-CTLA4-Ig+AAV8-CD40-Ig groups have more GFP expression than the AAV8-CTLA4-Ig group. These results indicate that immunosuppressive treatment with CTLA4-Ig and CD40-Ig can better improve transgene expression after repeated administration of AAV8 vectors.

In addition, exogenous transgene expression was monitored dynamically by AAV injection using AAV8-GLUC (fig. 4C). Mice were preimmunized with AAV8-CTLA4-Ig or a combination of AAV8-CTLA4-Ig and AAV8-CD40-Ig, and AAV8-GFP was used for the control group of HD and MD. After 4 weeks, mice were given two injections 6×10 intravenously every 3 weeks ¹¹ vg/kg AAV8-GLUC. In the AAV8-CTLA4 and AAV8-ctla4+ AAV-CD40 groups, GLUC expression was minimal at the first injection of AAV8-GLUC, but significantly increased at the second injection, above control levels (fig. 4D) (5 weeks, ×p<0.001). This result is consistent with the findings shown in fig. 2B, i.e., AAV immunity transiently proliferated with the first AAV8-GFP administration following administration, whereas AAV immune tolerance developed upon the second AAV8-GFP injection. Although at 6X 10 ¹¹ vg/kg single doseGLUC expression in naive (naive) mice injected in amounts with AAV8-GLUC was approximately five times the level of expression upon re-administration (fig. 4E), but sustained expression of CTLA4-Ig and CTLA4-ig+cd40-Ig significantly improved exogenous gene expression after repeated systemic administration of AAV vectors compared to the group without immunosuppressive treatment.

This study clearly shows that successful delivery of AAV8 expressing CTLA4-Ig and CD40-Ig induces immune tolerance that specifically inhibits immune responses against AAV8, which allows repeated administration of AAV8 expressing another transgene of interest, such as GFP or GLUC. It is well known in the art that GFP and GLUC are two well-known model genes that serve the purpose of providing proof of concept only. In other words, if GFP and GLUC are found to be expressed in the present invention, other genes of interest, such as those having therapeutic potential or application, may also be expressed in the same or similar manner. It is expected that one skilled in the art could easily replace GFP and GLUC in the examples with other therapeutic genes and achieve the desired results.

Continuous expression of CTLA4-Ig and CD40-Ig does not affect humoral immunity against AAV843

Although CTLA4-Ig and CD40-Ig inhibit AAV8 NAb, it is unclear whether they can inhibit NAb production from other serotypes. To verify this idea, it was tested whether Nab of AAV843 could be inhibited.

Two groups of mice were injected intravenously with two different doses (HD and MD) of AAV8-CTLA4-Ig or AAV8-CTLA4-ig+aav8-CD40-Ig in week 0, followed by two administrations of AAV8-GFP vector at week 8 (PFAW 8) and week 15 (PFAW 15), respectively (see fig. 5A). To verify long-term expression of CTLA4-Ig or CTLA4-Ig and CD40-Ig to induce antigen-specific tolerance of other AAV vectors, xiang Xiao mice were injected intravenously with AAV843-GLUC at week 20, a novel AAV serotype that delivered gaussian luciferase (fig. 5A).

No NAb titers of AAV843 were detectable in all groups prior to treatment with AAV843-GLUC, and AAV8 NAb titers in all CTLA 4-Ig-expressing groups remained negative after PFAW 16 (fig. 5B). At PFAW 20, AAV843 was injected into mice pre-treated with AAV8-CTLA4-ig+aav8-CD40-Ig or AAV8-CTLA4-Ig, and NAb titers of AAV843 increased to a high level of over 1:32 after one week, while transgenic GLUC expression was severely inhibited (fig. 5C). Thereafter, NAb levels gradually decreased (fig. 5B, < p <0.05, < p < 0.01), and GLUC expression was up-regulated (fig. 5C). The AAV8-CTLA4-Ig+AAV8-CD40-Ig HD group had the highest GLUC expression (FIG. 5C, < 0.05). The results show that delivery of AAV8-CTLA4-Ig or AAV8-CTLA4-ig+aav8-CD40-Ig allows for prolonged expression of exogenous CTLA4-Ig and CD40-Ig in vivo, which not only provides antigen-specific tolerance of AAV8, but also effectively reduces antibodies to AAV 843. Interestingly, immune tolerance to AAV8 also applies to AAV843, as demonstrated by the gradual decrease in Nab levels of AAV843 and stable GLUC expression of AAV 843.

Long-term expression of CTLA4-Ig and CD40-Ig inhibits T cell activation and B cell proliferation

To elucidate in depth the effect of long-term expression of CTLA4-Ig and CD40-Ig on immune responses, subsets of T cells and B cells were analyzed using flow cytometry. CD4 in Peripheral Blood Mononuclear Cells (PBMC) at PFAW 2 ⁺ T cells and B220 ⁺ CD19 ⁺ B cell frequency was not significantly different between immunosuppressant and control groups (fig. 6A). In contrast to the AAV8-GFP group, AAV8-CTLA4-Ig and AAV8-CTLA4-Ig+AAV8-CD 40-CD 25 in the Ig group ⁺ FoxP3 ⁺ Regulatory T cells (tregs) decreased at PFAW 2 and 7 (fig. 6B). Tregs in HD group have lower frequency (×p)<0.01). At PFAW 17, CD4 from PBMC was explored under repeated administration of AAV8-GFP ⁺ GL7 ⁺ T cell ratio. GL 7-expressing CD4 in AAV8-CTLA4-Ig, AAV8-CD40-Ig HD, and AAV8-CTLA4-Ig+AAV8-CD40-Ig groups, as compared to control groups ⁺ There was a slight decrease in T cell frequency, but these changes were not statistically significant, and GL7 was an activation marker expressed on hair-center T cells (fig. 6C and 6D).

At PFAW 25, CD4 in spleen was studied ⁺ T cells, B cells, and tregs. CD4 in the AAV8-CTLA4-Ig+AAV8-CD40-Ig HD group compared to the AAV8-GFP HD group ⁺ Frequency of T cells was decreased (FIGS. 6E-6F, & p <0.05). AAV8-CTLA4-Ig HD (×p) compared to control group<0.01)、AAV8-CTLA4-Ig MD(*p<0.05)、AAV8-CD40-Ig HD(**p<0.01 AAV8-CTLA4-ig+aav8-CD40-Ig HD (×p)<0.01 Spleen tregs in group were reduced, indicating that immunosuppressant treatment affected Treg subpopulations in a dose-dependent manner. B220 in spleen ⁺ CD19 ⁺ B cells did not significantly differ between immunosuppressant and control groups (fig. 6E-6F), but tendencies were reduced for AAV8-CTLA4-Ig HD group (p=0.041) and AAV8-CTLA4-ig+aav8-CD40-Ig HD group (p=0.033) compared to the tendencies previously at PFAW 2 (fig. 6G). The results indicate that CTLA4-Ig and CD40-Ig suppress immune responses against AAV by inhibiting T cell activation and B cell proliferation.

Discussion of the invention

rAAV is widely used in gene therapy as a promising gene vector. However, its use is limited by the widespread presence of nabs and macrophages against rAAV, and up to 70% of the population has nabs (Mingozzi F et al, 2013). Patients with high NAb titers cannot be treated with rAAV gene drugs, and currently, rAAV gene therapy is also a monotherapy technique, because NAb resulting from the first treatment causes rAAV inactivation, and then fails to function upon re-administration. NAb titers of 1:5 were reported to almost prevent human FIX expression of rAAV delivery in non-human primates (Jiang H et al, 2006). In addition, cytotoxic T cells also reduce transgene products by depleting AAV-infected cells (Murphy SL et al, 2008). Thus, there is an urgent need for safe and effective clinical strategies for suppressing AAV immune responses to achieve stable transgene expression and vector re-administration.

In this embodiment, CTLA4-Ig or CD40-Ig is expressed by in vivo AAV8 delivery. Inhibition and immune tolerance of NAb was analyzed when AAV8-GFP or AAV843-GLUC was administered as an immunogen. CTLA4-Ig or CD40-Ig can be continuously expressed and effectively secreted into serum after single intravenous injection of AAV8-CTLA4-Ig or AAV8-CD 40-Ig. Previous studies have shown that exogenous CTLA4-Ig or anti-CD 40L antibodies block the humoral immunity of rAAV and that rAAV re-administration was successfully achieved via pulmonary or intramuscular injection (Halbert CL et al, 1998 and Lorain, 2008). Intravenous delivery of rAAV gene drugs is the safest and convenient, but NAb may be easily neutralized by AAV (Wang D et al, 2019). It was found that although NAb was strongly up-regulated after the first administration of AAV8 immunogen, NAb was rapidly decreased thereafter. Interestingly, NAb was not significantly upregulated following secondary administration of AAV8 immunogen in either high dose CTLA4-Ig or CTLA4-Ig+CD40-Ig groups. In addition, long-term expression of CTLA4-Ig or CTLA4-Ig and CD40-Ig also reduces anti-AAV IgG after multiple intravenous administrations of the same AAV vector. Further, cytokine levels have been shown to be unaffected after long-term expression of CTLA4-Ig and CD 40-Ig. These results indicate that CTLA4-Ig and CTLA4-ig+cd40-Ig inhibited NAb in a dose-dependent manner and gradually induced immune tolerance to AAV 8. Furthermore, long-term expression of CTLA4-Ig and CD40-Ig in vivo does not deplete humoral immunity, suggesting that it is a safe and effective candidate for establishing immune tolerance against AAV.

It was observed that expression of exogenous genes, GFP or GLUC was improved when CTLA4-Ig and CD40-Ig were expressed chronically. Meanwhile, when CTLA4-Ig and CD40-Ig are co-administered, the exogenous gene is strongly expressed, compared to single administration of CTLA 4-Ig. Several injections of CTLA4-ig+mr1 were reported to preserve but not improve exogenous gene expression when re-administered rAAV (Halbert CL et al, 1998 and Lorain, 2008). However, in this example, no improvement in GFP expression was observed upon a single administration of CD 40-Ig. Previously, moskalkoko et al demonstrated that anti-CD 40L antibodies failed to improve transgene expression after systemic re-administration of AAV2 vector (moskalkoko M et al, 2000), and CTLA4-Ig was thought to play a dominant role in immunomodulation when co-expressing CTLA4-Ig and MR1 (Manning WC et al, 1998). Notably, upon detection of another transgene product, GLUC, delivered by AAV843 after two AAV8-GFP administrations, GLUC expression was significantly inhibited and NAb against AAV843 increased sharply immediately. Thereafter, AAV843 NAb titers decreased and GLUC expression was upregulated, indicating that sustained expression of CTLA4-Ig and CD40-Ig again inhibited anti-AAV 843 NAb. The results indicate that the immune tolerance induced by CTLA4-Ig and CD40-Ig is antigen-selective and specific for AAV8, but that this tolerance is also cross-reactive for different serotypes (i.e., AAV 843).

CTLA4-Ig and CD40-Ig via blocking CD28/B7 and CD40/CD40L co-stimulatory pathwaysRegulatory T cells and B cells (Boise LH et al, 1995 and June CH et al, 1990). In this example, CD4 in the high dose AAV8-CTLA4-Ig+AAV8-CD40-Ig group ⁺ T cells, CD4 ⁺ GL7 ⁺ T cells and B220 ⁺ CD19 ⁺ B-cell depletion indicates that T-cell activation and B-cell proliferation are inhibited. The results also demonstrate that CTLA4-Ig+CD40-Ig has better inhibition of antibody production and transgene expression improvement over multiple AAV administrations. However, the mechanism by which CTLA4-Ig and CD40-Ig induce tolerance to AAV is still unclear. Several studies have shown tolerance induction associated with Treg expansion (Sakaguchi S et al, 2001 and Bacchetta R et al, 2005). Surprisingly, however, in this example, the proportion of tregs in the CTLA 4-Ig-engaging group is reduced. Vogel et al demonstrated that Treg expansion was observed in the presence of MR1 alone or MR1 in combination with low dose CTLA4-Ig (Vogel I et al 2016). However, treg expansion was inhibited when CTLA4-Ig doses were equal to or greater than about 50 μg/ml, indicating that high doses of CTLA4-Ig provided immunosuppression in a Treg-independent manner (Vogel I et al 2016). In this example, the peak concentration of CTLA4-Ig in the CTLA 4-Ig-engaging group reaches about 200-600 μg/ml, corresponding to a high dose of soluble protein CTLA4-Ig, thereby down-regulating the Treg ratio.

In summary, sustained expression of CTLA4-Ig and CD40-Ig carried by certain serotypes of AAV can effectively suppress this AAV-induced immunity, establish immune tolerance to this AAV vector, and improve transgene expression following repeated intravenous administration of the same serotypes of AAV. In addition, sustained expression of these two immunosuppressants does not affect overall humoral immunity. These results may provide safe and convenient use for patients with NAb or who need to administer AAV gene drugs clinically multiple times.

Reference is made to:

altschul SF et al, basic local alignment search tool (Basic local alignment search tool), "journal of molecular biology (J.mol. Biol.), (1990) 215:403-410.

Altschul SF et al, with gaps BLAST and PSI-BLAST: new generation protein database search programs (Gapped BLAST and PSI-BLAST: a new generation of protein database search programs), "Nucleic Acids research (Nucleic Acids Res.), (1997) 25:3389-3402).

Higgins DG et al, using CLUSTAL for multiple sequence alignment (Using CLUSTAL for multiple sequence alignments), "Methods of enzymology" (1996) 266:383-402.

Larkin MA et al, clustal W and Clustal X version 2.0 (Clustal W and Clustal X version 2.0) & Bioinformatics (Oxford, england) 23 (21): 2947-8.

Goode I et al, regulatory B cells: new "it" cells (Regulatory B cells: the new "it" cells), "transplantation procedure (Transplant Proc), (2014) 46:3-8.

Good NE et al, hydrogen ion buffer for biological research (Hydrogen ion buffers for biological research) & Biochemistry (1966) 5,2,467-477.

Wu GY et al, receptor-mediated in vitro Gene conversion by soluble DNA vector System (Receptor-mediated in vitro gene transformation by a soluble DNA carrier system) & journal of biochemistry (J Biol Chem) (1987) 262 (10): 4429-32.

CpG motifs in Krieg AM et al bacterial DNA trigger direct B cell activation (CpG motifs in bacterial DNA trigger direct B-cell activation) & Nature (1995) 374:546-549.

Yamamoto S et al require the synthesis of unique palindromic sequences in the oligonucleotide to induce IFN and enhance IFN-mediated natural killer activity (Unique palindromic sequences in synthetic oligonucleotides are required to induce IFN and augment IFN-mediated natural killer activity) J.Immunol.1992.148:4072-76.

Ballas ZK et al, induces NK activity in mouse and human cells through oligodeoxynucleotides and CpG motifs in bacterial DNA (Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA) & J.Immunol.1996.157:1840-45.

Kabat EA et al, protein sequence of immunological significance (Sequences of Proteins of Immunological Interest), 5 th edition, besseda national institutes of health, malyland (National Institutes of Health, bethesda, MD) (1991).

Contribution of CpG motifs to the immunogenicity of DNA vaccines by Klinman DM et al (Contribution of CpG motifs to the immunogenicity of DNAvaccines) J.Immunol.1997.158:3635-39.

Sato Y et al, immune stimulating DNA sequences necessary for effective intradermal gene immunization (Immunostimulatory DNA sequences necessary for effective intradermal gene immunization) Science (1996) 273:352-354.

Pisetsky DS, immunological Properties of DNA (The immunologic properties of DNA) J.Immunol.1996.156:421-423.

Shimada S et al, enhanced natural killer cell activity in vivo with the deoxyribonucleic acid fraction of BCG (In vivo augmentation of natural killer cell activity with a deoxyribonucleic acid fraction of BCG), "J.J.cancer Res.) (1986) 77:808-816.

Cowdery JS et al, bacterial DNA induces NK cells to produce IFN-gamma in vivo and increases lipopolysaccharide toxicity (Bacterial DNAinduces NK cells to produce IFN-gamma in vivo and increases the toxicity of lipopolysaccharides) & journal of immunology (1996) 156:4570-75.

Roman H et al immunostimulatory DNA sequences function as T helper-1 promoting adjuvants (Immunostimulatory DNAsequences function as T helper-1-promoting adjuvants) & Nature medicine (Nat. Med.) & lt 1997) 3:849-854.

Lipford GB et al, synthetic oligonucleotides containing CpG promote B cell and cytotoxic T cell responses to protein antigens: a new class of vaccine adjuvants (CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants), "European journal of immunology (Eur. J. Immunol.), (1997) 27:2340-44.

Wang D et al, adeno-associated viral vector (Adeno-associated virus vector as a platform for gene therapy delivery) as a delivery platform for gene therapy, nature comment drug discovery (Nature Reviews Drug Discovery) (2019) 18 (5): 358-378.

Inhibition of experimental allergic encephalomyelitis by Arima T et al CTLA4Ig (Inhibition by CTLA4Ig of experimental allergic encephalomyelitis) & journal of immunology (1996) 156:4916-24.

Li W et al CTLA4Ig gene transfer moderates mouse abortions by amplifying CD4+CD25+ regulatory T cells and inducing indoleamine 2,3-dioxygenase (CTLA 4Ig gene transfer alleviates abortion in mice by expanding CD4+CD25+ regulatory T cells and inducing indoleamine 2, 3-dioxygenase), (J Reproductive Immunology), 2009) 80 (1-2): 1-11.

Dall' Era M et al CTLA4Ig: a novel costimulatory inhibitor (CTLA 4Ig: a novel inhibitor of costimulation) is disclosed in Lupus (Lupus) (2004) 13 (5) 372-376.

Slavik JM et al CD28/CTLA-4and CD80/CD86 families: signal transduction and function (CD 28/CTLA-4and CD80/CD86 family: signaling and function) & immunology research (Immunol Res) & 1999, 19:1-24.

Genovese MC et al, abamectin (Abatacept for rheumatoid arthritis refractory to tumor necrosis factor alpha inhibition) for inhibition of rheumatoid arthritis refractory tumor necrosis factor alpha (New England medical journal (N.Engl. J. Med.) (2005), 353:1114-1123.

Charpentier B. Beracep: a novel immunosuppressant for kidney transplant recipients (Belatacept: a novel immunosuppressive agent for kidney transplant recipients) review of clinical immunology experts (Expert Rev. Clin. Immunol.) (2012) 8:719-728.

The 39-kDa protein on Noelle RJ et al activated helper T cells binds to CD40 and transduces a B cell homology activation signal (A39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells) Proc Natl Acad Sci (1992) 89 (14): 6550-4.

Short-term treatment of Boumpas DT et al BG9588 (anti-CD 40 ligand antibody) improved the serological activity and decreased haematuria (Ashort course of BG9588 (anti-CD 40 ligand anti) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis) in patients with proliferative lupus glomerulonephritis (2003) 48 (3): 719-727.

Kirk AD et al CTLA4Ig and anti-CD40 ligand to prevent renal allograft rejection in primates (CTLA 4Ig and anti-CD40 ligand prevent renal allograft rejection in primates) Proc. Natl. Acad. Sci. USA (1997) 94:8789-8794.

Successful re-administration of adeno-associated viral vectors to the lungs of mice by Halbert CL et al requires transient immunosuppression during initial exposure (Successful readministration of adeno-associated virus vectors to the mouse lung requires transient immunosuppression during the initial exposure) & journal of Virology (1998) 72 (12): 9795-805.

Lorain et al transient immunomodulation allowed repeated injections of AAV1 and correction of muscular dystrophy in multiple muscles (Transient immunomodulation allows repeated injections of AAV1 and correction of muscular Dystrophy in multiple muscles) & molecular therapy (Mol Ther) & 2008,16 (3): 541-547.

Mcintosh JH et al, AAV mediated Gene transfer, successfully attenuated humoral immunity to viral capsids and transgenic proteins with non-depleting CD4 antibodies and cyclosporin (Successful attenuation ofhumoral immunity to viral capsid and transgenic protein following AAV-mediated Gene transfer with a non-depleting CD4 antibody and cyclosporine), (Gene Ther), (2012) 19 (1): 78-85.

Sustained muscle expression of dystrophin from high capacity adenovirus vectors by T cell co-stimulation of blocked systemic gene transfer by Jiang Z et al (Sustained muscle expression of Dystrophin from a high-capacity adenoviral vector with systemic gene transfer of T Cell costimulatory blockade), (2004) 10 (4): 688-696.

Chirmule N et al re-administer adenovirus vectors in the lungs of non-human primates by blocking CD40-CD40 ligand interactions (Readministration of adenovirus vector in nonhuman primate lungs by blockade of CD-CD 40 ligand interactions), (2000) 74 (7): 3345-52).

Transient disruption of Yang Y et al CD40 ligand function reduces the immune response to adenovirus vectors in mouse liver and lung tissue (Transient subversion of CD, ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung tissues) & journal of virology (1996 a) 70:6370-6377.

Yang Y et al CD40 ligand-dependent T cell activation: B7-CD28 signaling by CD40 is required (CD 40 ligand-dependent T cell activation: requirement of B7-CD28 signaling through CD 40) science (1996B) 273:1862-1864.

YeX et al prevent and reverse lupus (Prevention and reversal of lupus in NZB/NZW mice by costimulatory blockade with adeno-associated viruses-mediated gene transfer) in NZB/NZW mice by co-stimulatory blockade of adeno-associated virus mediated gene transfer, (2005) 52 (12): 3975-3986.

Adeno-associated viral vectors carrying human micro-dystrophin gene from Wang B et al are effective in ameliorating muscular dystrophy in mdx mouse models (Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model) Proc. Natl. Acad. Sci. USA, (2000) 97 (25): 13714-13719.

Bakhos A et al, gauss luciferase reporter assay (Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo) for monitoring culture and in vivo biological processes (2009) Nature protocols 4 (4): 582-91.

Feasibility and safety of systemic rAAV9-hNAGLU delivery by Murrey DA et al for the treatment of mucopolysaccharidosis IIIB: toxicology, biodistribution and immunological assessment of primate (Feasibility and safety of systemic rAAV-hNAGLU delivery for treating mucopolysaccharidosis IIIB: toxicology, biodistribution, and immunological assessments in primates) & clinical development of human gene therapy (hum. Gene Ther. Clin. Dev.) & gt (2014) 25,72-84.

Prevalence of immune responses against adeno-associated viruses in the international healthy donor cohort of Kruzik A et al (Prevalence of anti-adeno-associated virus immune responses in international cohorts of healthy donors) & molecular therapy method clinical development (Mol Ther Methods Clin Dev.) & gt (2019) 14:126-133.

Immune response of Mingozzi F et al to AAV vectors: overcoming the obstacle to successful gene therapy (Immune responses to AAV vectors: overcoming barriers to successful gene therapy) & Blood (2013) 122 (1) & 23-26.

The effect of transient immunosuppression by Jiang H et al on rhesus monkey adeno-associated virus-mediated liver-directed gene transfer and on human gene therapy (Effects of transient immunosuppression on adeno associated virus-mediated, river-directed gene transfer in rhesus macaques and implications for human gene therapy), (2006) 108:3321-3328.

Long-term susceptibility of Murphy SL et al to antibody-mediated neutralization by liver-targeted adeno-associated vectors (Prolonged susceptibility to antibody-mediated neutralization for adeno-associated vectors targeted to the liver), (2008) 16 (1): 138-145).

Epitope mapping of human neutralizing antibodies against adeno-associated virus type 2 by Moskalenko M et al: effects on Gene therapy and viral Structure (Epitope mapping of human anti-adeno-associated virus type 2neutralizing antibodies:implications for gene therapy and virus structure), "J.Virol.2000: (74) 1761-1766).

Manning WC et al transient immunosuppression allows transgene expression after re-administration of adeno-associated viral vectors (Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors) & human gene therapy (hum. Gene Ther.) & 1998) 9,477-485.

Boise LH et al CD28 co-stimulation can promote T cell survival by enhancing Bcl-XL expression (CD 28costimulation can promote T cell survival by enhancing the expression of Bcl-XL) & immunity (immunity.) & lt1995 & gt 3,87-98.

Role of June CH et al CD28 receptor in T cell activation (rule of the CD28 receptor in T-cell activation) & lt immunology today (immunol. Today.) & lt (1990) 11,211-216.

Sakaguchi S et al cd25+cd4+ regulatory T cells maintain immune tolerance: their combined action in controlling autoimmunity, tumor immunity and graft tolerance (Immunologic tolerance maintained by CD25+CD4+ regulatory T cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance) reviewed in immunology (immunol. Rev.) (2001) 182,18-32.

Bacchetta R et al cd4+ regulatory T cells: induction mechanism and effector function (CD4+ regulatory T cells: mechanisms of induction and effector function), "review of autoimmune forces (Auto immun. Rev.), (2005) 4,491-496.

Regulatory T cell dependent and independent immunosuppressive mechanisms by Vogel I et al (Regulatory T cell-dependent and independent mechanisms of immune suppression by CD28/B7 and CD40/CD40L costimulation blockade) through CD28/B7 and CD40/CD40L co-stimulatory blockade, (2016) 197:533-540.

Najafian N et al CTLA4-Ig: a novel immunosuppressive agent (CTLA 4-Ig: a novel immunosuppressive agent) is described in review of pharmaceutical specialists (Expert Opin Investig Drugs) (2000) 9:2147-57.

Puttaraju M et al, spliceosome-mediated RNA trans-splicing as a tool for gene therapy (Spliceosome-mediated RNA trans-splicing as a tool for gene therapy) & Nature Biotech (1999) 17:246.

Sharp PA et al, molecular biology (Molecular biology), RNA interference (RNA interference), science (2000) 287:2431.

Gorman L et al, modified U7 microRNA for stable modification of pre-mRNA splice patterns (Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs) Programming, proc. Natl. Acad. Sci. USA (1998) 95:4929.

Adli M et al, CRISPR kits for genome editing and others (The CRISPR tool kit for genome editing and beyond) & lt, natural communication (Nat Commun.) & lt 2018 & gt 9 (1): 1911.

Sambrook and russell, molecular cloning (Molecular Cloning), 3 rd edition, cold spring harbor laboratory press (Cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y.), (2001).

Ausubel FM et al Current protocols in molecular biology (Current Protocols in Molecular Biology), john Wiley father-son Press (John Wiley & Sons) (1992).

Ausubel FM et al, current guidelines for molecular biology experiments (Current Protocols in Molecular Biology), green publishing Association (Greene Publishing Associates) and John Willi's father, N.Y. (John Wiley & Sons, new York, N.Y.), 1994.

Sambrook J et al, molecular cloning: laboratory Manual (Molecular Cloning, a Laboratory Manual), 2 nd edition, cold spring harbor Press (1989) of Cold spring harbor, new York.

Laughlin CA et al, cloning of infectious adeno-associated viral genome in bacterial plasmids (Cloning of infectious adeno-associated virus genomes in bacterial plasmids) & lt, gene & gt (1983) 23 (1): 65-73 (1983).

BERNARD NF et al, field Virology, 4 th edition, 2 (69), lippincott-Raven Publishers (2007).

GaoG et al, clades of Adeno-associated viruses spread widely in human tissue (Clades of Adeno-associated viruses are widely disseminated in human tissues) & journal of virology (2004) 78:6381-6388.

Kotin RM et al, adeno-associated virus as a vector for human gene therapy (Prospects for the use of adeno-associated virus as a vector for human gene therapy) & lt, human gene therapy (1994) 5:793-801.

Berns, KI. parvoviridae and replication thereof (Parvoviridae and their Replication) & basic virology (Fundamental Virology), 2 nd edition, B.N. fields and D.M. Knipe, editions (1990).

Characterization of adeno-associated virus (AAV) DNA replication and integration (Characterization of Adeno-associated virus (AAV) DNA replication and integration), doctor article, university of Pittsburgh, pa. (1996).

Naso MF et al, adeno-Associated Virus (AAV) as a vector for gene therapy (Adeno-Associated Virus (AAV) as a Vector for Gene Therapy) biopharmaceutical (Biodrugs) 31 (4): 317-334.

Quantitative analysis of the packaging ability of recombinant adeno-associated virus by Dong JY et al (Quantitative analysis of the packaging capacity of recombinant adeno-associated virus) & lt, human Gene therapy (1996) 7 (17): 2101-12.

Chamberlain K et al, express transgenes that exceed the packaging capacity of adeno-associated viral capsids (Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids) & method of human gene therapy (Hum Gene Ther Methods) & lt 2016) 27 (1): 1-12.

Carter BJ, adeno-associated viral vector (Adeno-associated virus vectors) Current evaluation of biotechnology (Current Opinion in Biotechnology), 1992, 3:533-539.

Muzyczka N. adeno-associated virus is used as a universal transduction vector for mammalian cells (Use of adeno-associated virus as a general transduction vector for mammalian cells) & Current topics of microbiology and immunology (Curr. Top. Microbiol. Immunol.), (1992) 158:97-129.

Lebkowski JS et al adeno-associated virus: vector systems for efficient incorporation and integration of DNA into a variety of mammalian cell types (Adeno-associated viruses: a vector system for efficient introduction and integration of DNA into a variety of mammalian cell types) & molecular cell biology (1988) 8:3988-3996.

Vincent KA et al vaccine (Vaccines) (1990) volume 90, cold spring harbor laboratory Press.

Targeting integration of transfected and infected adeno-associated viral vectors containing neomycin resistance genes by Shelling AN and Smith MG. (Targeted integration of transfected and infected adeno-associated virus vectors containing the neomycin resistance gene) & lt, gene therapy (1994) 1:165-169.

Zhou SZ et al, adeno-associated virus 2 mediated efficient gene transfer into immature and mature subsets of hematopoietic progenitor cells of human cord blood (Adeno-associated virus 2-mediated high efficiency gene transfer into immature and mature subsets of hematopoietic progenitor cells in human umbilical cord blood), (J. Exp. Med.) (1994) 179:1867-1875).

Blacklow NR et al (1988) human Adeno-associated virus (Adeno-associated viruses of humans). In: pattern (eds.), "parvovirus and human disease (Parvoviruses and Human Disease),. CRC Press, boca Raton, FL) pages 165-174, bocalion, florida.

Rose J parvovirus propagation (Parvovirus reproduction) in Integrated virology (Comprehensive Virology), volume 3, pages 1-62. Edited by h.fraenkel-hart & r.r.wagner, new york: pruneme Press (Plenum Press) (1974).

Intracellular transport of recombinant adeno-associated viral vectors (Intracellular transport of recombinant adeno-associated virus vectors) by Nonnenmacher M et al (2012) 19 (6): 649-658).

Xu J. Et al, prevalence of neutralizing antibodies to AAV8, AAV9 and AAV843 in the Chinese population (Prevalence of neutralizing antibodies against AAV, AAV9, and AAV843 in a Chinese population) & International journal of clinical and laboratory medicine (Int J Clin Exp Med) & gt (2019) 12 (8): 10253-10261.

Fisher KJ et al, recombinant adeno-associated viral transduction for gene therapy is limited by leader synthesis (Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis) & journal of virology (1993) 70:520-532.

Graham FL et al, characterization of human cell lines transformed with DNA from human adenovirus type 5 (Characteristics of a human cell line transformed by DNA from human adenovirus type 5), "journal of general virology (J.Gen.Virol.), (1977) 36:59-72.

Activation of transgene expression in early region 4 of Brough DE et al is responsible for high levels of sustained transgene expression in adenovirus vectors in vivo (Activation of transgene expression by early region 4is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo), (1997) J.Virol.71:9206-9213.

Paolicali P et al, can effectively associate and deliver Surface modified PLGA-based nanoparticles of virus-like particles (Surface-modified PLGA-based nanoparticles that can efficiently associate and deliver virus-like particles) & Nanomedicine (2010) 5 (6): 843-853.

The Nanogel-based delivery of mycophenolic acid improves systemic lupus erythematosus (Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice) in mice (J.clinical research journal) 123 (4): 1741-1749.

Doubrow, microcapsules and nanoparticles in medicine and pharmacy (Microcapsules and Nanoparticles in Medicine and Pharmacy), CRC Press of Bokaraton (1992).

Mathiowitz E et al, polyanhydride microspheres as drug carrier I. Hot melt microencapsulation (Polyanhydride microspheres as drug carriers I. Hot-melt microencapsulation) & J. Control. Release (1987 a) 5:13-22.

Mathiowitz E et al, novel microcapsules for use in delivery systems (Novel microcapsules for delivery systems), "reactive Polymer (Reactive Polymers), (1987 b) 6:275-283.

Mathiowitz E et al, polyanhydride microspheres as drug carrier II. Microencapsulation by removal of solvent (Polyanhydride microspheres as drug carriers. II. Microencapsulation by solvent removal) & journal of applied Polymer science (J.appl. Polymer Sci.) & lt (1998) 35:755-744.

The synthesis and characterization of PLGA nanoparticles by Astete CE (Synthesis and characterization of PLGA nanoparticles), "J.Biomater. Sci. Polymer Edn, (2006) 17 (3): 247-289.

Avgoutakis K, pegylated poly (lactide) and poly (lactide-co-glycolide) nanoparticles: preparation, properties and possible applications in drug delivery (Pegylated poly (lactide) and poly (lactide-co-glycoide) nanoparticles: preparation, properties and possible applications in drug delivery) Current drug delivery (Current Drug Delivery) (2004) 1:321-333.

Reis CP et al, nanocapsule I. Preparation of polymer nanoparticles for drug delivery (nanoscaled I. Methods for preparation of drug-loaded polymeric nanoparticles) & lt/EN & gt, nanomedicine (2006) 2:8-21.

Sharpless VV et al, gradual huisgen cycloaddition procedure: copper (I) -catalyzed regioselective "ligation" of azides and terminal alkynes (A stepwise huisgen cycloaddition process: coater (I) -catalyzed regioselective "ligation" of azides and terminal alkynes) German International edition of applied chemistry (Angew. Chem. Int. Ed.) (2002) 41 (14): 2596-2599.

Meldal M et al, copper-catalyzed azide-alkyne cycloaddition (Cu-catalyzed azide-alkyne cycloaddition), "chemical review (chem. Rev.), (2008) 108 (8): 2952-301.

Ito T et al, a convenient enzyme-linked immunosorbent assay for rapid screening of neutralizing antibodies against adeno-associated virus (Aconvenient enzyme-linked immunosorbent assay for rapid screening of anti-adeno-associated virus neutralizing antibodies) & lt (Ann Clin biochem.) & lt 46 (Pt 6): 508-510).

Beattie WG et al, deduced from the cloned cDNA partial sequence, the structure and evolution of human alpha fetoprotein (Structure and evolution of human alpha-fetoprotein deduced from partial sequence of cloned cDNA) & lt, gene (1982) 20:415-422.

Lichenstein et al, afatim, are novel members of the albumin, alpha-fetoprotein and vitamin D binding protein gene families (Afamin is a new member of the albumin, alpha-fetoprotein, and vitamin D-binding protein gene family) & journal of biochemistry (1984) 269:18149-18154.

Cooke NE et al Serum vitamin D binding protein is the third member of the albumin and alpha fetoprotein gene family (Serum vitamin D-binding protein is a third member of the albumin and alpha fetoprotein gene family) & journal of clinical research (1985) 76:2420-2424.

Mueller DL et al, cloning amplification and functional cloning inactivation: the co-stimulatory signaling pathway determines the outcome of T cell antigen receptor occupancy (Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy) & immunology annual review (Annu Rev. Immunol.) & 1989) 7:445-480.

Bessis N et al immune response to Gene therapy vectors: effects on vector function and effector mechanisms (Immune responses to gene therapy vectors: influenceon vector function and effector mechanisms), "Gene therapy (2004) 11:S10-S17.

Jones NH et al (Isolation of complementary DNA clones encoding the human lymphocyte glycoprotein T/Leu-1) isolation of complementary DNA clones encoding human lymphocyte glycoprotein T1/Leu-1 (1986) 323:346-349.

Elgueta R et al, immune system, CD40/CD40L, involved molecular mechanisms and functions (Molecular mechanism and function of CD/CD 40L engagement in the immune system) & immunology review (2009) 229 (1).

Clark EA et al, proc.Natl.Acad.Sci.USA (1986) 83 (12): 4494-8), mediated activation of human B cells by two different cell surface differentiation antigens Bp35 and Bp50 (Activation of human B cells mediated through two distinct cell surface differentiation antigens, bp35 and Bp 50).

The 39-kDa protein on activated helper T cells of Noelle RJ et al binds to CD40 and transduces a cognate activation signal for B cells (Proc. Natl. Acad. Sci. USA (1992) 89 (14): 6550-4).

Datta-Mannan A et al, monoclonal antibody clearance modulating the effects of IgG interactions with neonatal Fc receptor (Monoclonal antibody clearance. Image of modulating the interaction of IgG with the neonatal Fc receptor) journal of biochemistry (2007) 282 (3): 1709-17.

FcRn Affinity-pharmacokinetics relationship of five human IgG4 antibodies engineered for improved in vitro FcRn binding characteristics in cynomolgus monkeys (FcRn Affinity-Pharmacokinetic Relationship of Five Human IgG4 Antibodies Engineered for Improved In Vitro FcRn Binding Properties in Cynomolgus Monkeys) Datta-Mannan A et al (Drug Metabolism and Disposition) 2012) 40 (8) 1545-1555.

Hinton PR et al, engineered human IgG1 antibodies with longer serum half-lives (An Engineered Human IgG1 Antibody with Longer Serum Half-Life) J.Immunol.2006) 176:346-356.

Dall' Acqua WF et al, properties of human IgG1 engineered for enhanced binding to neonatal Fc receptor (FcRn) (Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn))journalof biochemistry (2006) 281 (33): 23514-24.

Zalevsky J et al, enhanced antibody half-life increased in vivo activity (Enhanced antibody half-life improves in vivo activity), "Nat Biotechnology (Nat Biotechnol.) (2010) 28 (2): 157-159.

Pharmacokinetics of humanized monoclonal anti-tumor necrosis factor- { alpha } antibodies and neonatal Fc receptor variants in mice and cynomolgus monkeys (Pharmacokinetics of humanized monoclonal anti-tumor necrosis factor- { alpha } antibody and its neonatal Fc receptor variants in mice and cynomolgus monkeys) Deng R et al, drug metabolism and treatment (2010) 38 (4): 600-5.

Maeda a et al, identified human IgG1 variants with enhanced FcRn binding but not increased binding to rheumatoid factor autoantibodies (Identification of human IgG1 variant with enhanced FcRn binding and without increased binding to rheumatoid factor autoantibody) & monoclonal antibodies (MAbs) & 2017 & 9 (5): 844-853.

Ha JH et al, immunoglobulin Fc heterodimer platform technology: therapeutic antibodies and proteins range from design to use (Immunoglobulin Fc Heterodimer Platform Technology: from Design to Applications in Therapeutic Antibodies and Proteins) & Front immunological protocol (2016) 7:394.

Wei H et al, structural basis for novel heterodimeric Fc for bispecific antibody production (Structural basis of a novel heterodimeric Fc for bispecific antibody production) & lt & gtcancer target (Oncotarget) & lt.2017 & gt 8 (31): 51037-51049.

Atwell S et al use phage display libraries to remodel stable heterodimers of the domain interface of homodimers (Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library) & journal of molecular biology (1997) 270 (1): 26-35.

Von Kreudenstein TS et al improve the biophysical properties of bispecific antibody scaffolds to aid in developability: mass by molecular design (Improving biophysical properties of a bispecific antibody scaffold to aid developability: quality by molecular design) monoclonal antibody (2013) 5 (5): 646-54.

Gunasekaran K et al enhance antibody Fc heterodimer formation by electrostatic steering effects: use in bispecific molecules and monovalent IgG (Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG) & journal of biochemistry (2010) 285 (25): 19637-46.

Choi HJ et al, heterodimer Fc-based bispecific antibodies targeting both VEGFR-2and Met, demonstrate potent antitumor activity (A heterodimeric Fc-based bispecific antibody simultaneously targeting VEGFR-2and Met exhibits potent antitumor activity) & molecular Cancer therapy (Mol Cancer Ther.) & gt (2013) 12 (12): 2748-59.

Klein C et al, progress in overcoming the chain association problem in bispecific heterodimeric IgG antibodies (Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies) & monoclonal antibodies (2012) 4 (6) & 653-63.

Xu Z et al, CTLA4-Ig affinity and cross-reactivity was engineered to modulate T cell co-stimulation (Affinity and Cross-Reactivity Engineering of CTLA4-Ig To Modulate T Cell Costimulation) & journal of immunology (2012) 189 (9) 4470-4477.

Xu H et al, nanocarriers in gene therapy: overview (Nanocarriers in gene therapy: a review) & journal of biomedical nanotechnology (J Biomed nanotechnol.) & lt (2014) 10 (12) & lt 3483-507 & gt.

Recent advances in nanomaterials for gene delivery, reviewed in Riley MK et al (Recent Advances in Nanomaterials for Gene Delivery-a Review) & nanomaterials (bassell) (Nanomaterials (Basel). & gt 2017) 7 (5): 94.

Sequence listing

<110> UNIVERSITY OF eastern China (EAST CHINA UNIVERSITY OF SCIENCE AND TECHNOLOGY)

<120> methods and kits for inducing immune tolerance to gene delivery targeting agents

<130> 075580-8004WO01, 075580-8004WO02

<150> PCT/CN2020/134283

<151> 2020-12-07

<160> 17

<170> patent In version 3.5

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<212> PRT

<213> artificial sequence

<220>

<223> full-length human CTLA-4 protein sequence with Signal peptide

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Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro

20 25 30

Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala

35 40 45

Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly

50 55 60

Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln

65 70 75 80

Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr

85 90 95

Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe

165 170 175

Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys

180 185 190

Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu

195 200 205

Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn

210 215 220

<210> 2

<211> 134

<212> PRT

<213> artificial sequence

<220>

<223> human CD28ECD

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Asn Lys Ile Leu Val Lys Gln Ser Pro Met Leu Val Ala Tyr Asp Asn

1 5 10 15

Ala Val Asn Leu Ser Cys Lys Tyr Ser Tyr Asn Leu Phe Ser Arg Glu

20 25 30

Phe Arg Ala Ser Leu His Lys Gly Leu Asp Ser Ala Val Glu Val Cys

35 40 45

Val Val Tyr Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr

50 55 60

Gly Phe Asn Cys Asp Gly Lys Leu Gly Asn Glu Ser Val Thr Phe Tyr

65 70 75 80

Leu Gln Asn Leu Tyr Val Asn Gln Thr Asp Ile Tyr Phe Cys Lys Ile

85 90 95

Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly

100 105 110

Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe

115 120 125

Pro Gly Pro Ser Lys Pro

130

<210> 3

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide-free human CTLA-4 extracellular Domain

<400> 3

Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile

1 5 10 15

Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val

20 25 30

Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys

35 40 45

Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser

50 55 60

Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln

65 70 75 80

Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu

85 90 95

Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile

100 105 110

Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp

115 120

<210> 4

<211> 173

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide-free human CD40 extracellular Domain

<400> 4

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 5

<211> 232

<212> PRT

<213> artificial sequence

<220>

<223> human Fc polypeptide sequence

<400> 5

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 6

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> human CTLA-4 Signal peptide sequences

<400> 6

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro

20 25 30

Val Phe Cys

35

<210> 7

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> human CD-40 Signal peptide sequence

<400> 7

Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Pro

20

<210> 8

<211> 397

<212> PRT

<213> artificial sequence

<220>

<223> mouse CTLA4-Ig complete sequence with Signal peptide

<400> 8

Met Ala Cys Leu Gly Leu Arg Arg Tyr Lys Ala Gln Leu Gln Leu Pro

1 5 10 15

Ser Arg Thr Trp Pro Phe Val Ala Leu Leu Thr Leu Leu Phe Ile Pro

20 25 30

Val Phe Ser Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala

35 40 45

Ser Ser His Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His

50 55 60

Asn Thr Asp Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln

65 70 75 80

Met Thr Glu Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly

85 90 95

Phe Leu Asp Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Leu Ile Ser Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro

165 170 175

Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile

180 185 190

Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile

195 200 205

Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln

210 215 220

Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln

225 230 235 240

Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu

245 250 255

Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys

260 265 270

Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys

275 280 285

Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro

290 295 300

Gln Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr

305 310 315 320

Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys

325 330 335

Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly

340 345 350

Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val

355 360 365

Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn

370 375 380

His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys

385 390 395

<210> 9

<211> 422

<212> PRT

<213> artificial sequence

<220>

<223> mouse CD40-Ig complete sequence with Signal peptide

<400> 9

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Leu Gly Gln Cys Val Thr Cys Ile Asp Lys Gln Tyr Leu

20 25 30

His Asp Gly Gln Cys Cys Asp Leu Cys Gln Pro Gly Ser Arg Leu Thr

35 40 45

Ser His Cys Thr Ala Leu Glu Lys Thr Gln Cys His Pro Cys Asp Ser

50 55 60

Gly Glu Phe Ser Ala Gln Trp Asn Arg Glu Ile Arg Cys His Gln His

65 70 75 80

Arg His Cys Glu Pro Asn Gln Gly Leu Arg Val Lys Lys Glu Gly Thr

85 90 95

Ala Glu Ser Asp Thr Val Cys Thr Cys Lys Glu Gly Gln His Cys Thr

100 105 110

Ser Lys Asp Cys Glu Ala Cys Ala Gln His Thr Pro Cys Ile Pro Gly

115 120 125

Phe Gly Val Met Glu Met Ala Thr Glu Thr Thr Asp Thr Val Cys His

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Gln Ser Ser Leu Phe Glu Lys

145 150 155 160

Cys Tyr Pro Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Val Leu Gln

165 170 175

Lys Gly Thr Ser Gln Thr Asn Val Ile Cys Gly Leu Lys Ser Arg Met

180 185 190

Arg Gly Thr Val Pro Arg Asp Cys Gly Cys Glu Pro Cys Ile Cys Thr

195 200 205

Val Pro Glu Val Ser Ser Val Ile Ile Phe Pro Pro Lys Pro Lys Asp

210 215 220

Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp

225 230 235 240

Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp

245 250 255

Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Leu Asn

260 265 270

Ser Ala Phe Arg Ser Val Gly Glu Leu Pro Ile Met His Gln Asp Trp

275 280 285

Leu Asn Gly Lys Glu Phe Lys Met Gln Gly Gln Gln Cys Ser Phe Pro

290 295 300

Cys Pro His Arg Glu Asn His Leu Pro Thr Pro Gln Lys Pro Lys Ala

305 310 315 320

Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp

325 330 335

Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile

340 345 350

Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn

355 360 365

Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys

370 375 380

Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys

385 390 395 400

Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu

405 410 415

Ser His Ser Pro Gly Lys

420

<210> 10

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> Oncoinhibin M Signal peptide

<400> 10

Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala

1 5 10 15

Leu Leu Phe Pro Ser Met Ala Ser Met Ala

20 25

<210> 11

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide-free human CTLA-4 extracellular Domain

<400> 11

Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg

1 5 10 15

Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu

35 40 45

Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp

50 55 60

Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp

115 120 125

<210> 12

<211> 223

<212> PRT

<213> artificial sequence

<220>

<223> mouse CTLA-4 full-length protein sequence

<400> 12

Met Ala Cys Leu Gly Leu Arg Arg Tyr Lys Ala Gln Leu Gln Leu Pro

1 5 10 15

Ser Arg Thr Trp Pro Phe Val Ala Leu Leu Thr Leu Leu Phe Ile Pro

20 25 30

Val Phe Ser Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala

35 40 45

Ser Ser His Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His

50 55 60

Asn Thr Asp Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln

65 70 75 80

Met Thr Glu Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly

85 90 95

Phe Leu Asp Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp Phe Leu Leu Trp Ile Leu Val Ala Val Ser Leu Gly Leu Phe Phe

165 170 175

Tyr Ser Phe Leu Val Ser Ala Val Ser Leu Ser Lys Met Leu Lys Lys

180 185 190

Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu

195 200 205

Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn

210 215 220

<210> 13

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> mouse CTLA-4 extracellular domain

<400> 13

Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His

1 5 10 15

Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu

35 40 45

Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp

50 55 60

Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp

115 120 125

<210> 14

<211> 174

<212> PRT

<213> artificial sequence

<220>

<223> mouse CD40 extracellular Domain

<400> 14

Leu Gly Gln Cys Val Thr Cys Ser Asp Lys Gln Tyr Leu His Asp Gly

1 5 10 15

Gln Cys Cys Asp Leu Cys Gln Pro Gly Ser Arg Leu Thr Ser His Cys

20 25 30

Thr Ala Leu Glu Lys Thr Gln Cys His Pro Cys Asp Ser Gly Glu Phe

35 40 45

Ser Ala Gln Trp Asn Arg Glu Ile Arg Cys His Gln His Arg His Cys

50 55 60

Glu Pro Asn Gln Gly Leu Arg Val Lys Lys Glu Gly Thr Ala Glu Ser

65 70 75 80

Asp Thr Val Cys Thr Cys Lys Glu Gly Gln His Cys Thr Ser Lys Asp

85 90 95

Cys Glu Ala Cys Ala Gln His Thr Pro Cys Ile Pro Gly Phe Gly Val

100 105 110

Met Glu Met Ala Thr Glu Thr Thr Asp Thr Val Cys His Pro Cys Pro

115 120 125

Val Gly Phe Phe Ser Asn Gln Ser Ser Leu Phe Glu Lys Cys Tyr Pro

130 135 140

Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Val Leu Gln Lys Gly Thr

145 150 155 160

Ser Gln Thr Asn Val Ile Cys Gly Leu Lys Ser Arg Met Arg

165 170

<210> 15

<211> 188

<212> PRT

<213> artificial sequence

<220>

<223> full-length human CTLA-4 protein sequence without signal peptide

<400> 15

Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg

1 5 10 15

Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu

35 40 45

Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp

50 55 60

Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Phe Leu

115 120 125

Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe Tyr Ser Phe

130 135 140

Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys Arg Ser Pro

145 150 155 160

Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu Pro Glu Cys

165 170 175

Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn

180 185

<210> 16

<211> 277

<212> PRT

<213> artificial sequence

<220>

<223> human CD40 full-length protein sequence with Signal peptide

<400> 16

Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu

20 25 30

Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val

35 40 45

Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

50 55 60

Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65 70 75 80

Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr

85 90 95

Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100 105 110

Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly

115 120 125

Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys

145 150 155 160

Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln

165 170 175

Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu

180 185 190

Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile

195 200 205

Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn

210 215 220

Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp

225 230 235 240

Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His

245 250 255

Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser

260 265 270

Val Gln Glu Arg Gln

275

<210> 17

<211> 257

<212> PRT

<213> artificial sequence

<220>

<223> human CD40 full-length protein sequence with Signal peptide

<400> 17

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg Ala Leu Val

165 170 175

Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile Leu Leu Val Leu

180 185 190

Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn Lys Ala Pro His

195 200 205

Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp Asp Leu Pro Gly

210 215 220

Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro

225 230 235 240

Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg

245 250 255

Gln

Claims

1. A method of inducing immune tolerance to an immunogenic target agent for gene delivery in a subject, the method comprising:

a) Administering an immunogenic inducer vehicle to the subject, wherein the immunogenic inducer vehicle comprises an inducer nucleic acid vector encoding at least one immunosuppressant, thereby allowing expression of the at least one immunosuppressant in the subject, the at least one immunosuppressant inhibiting an immune response in the subject against the inducer vehicle, wherein the immune response is cross-reactive with the target vehicle.

2. The method of claim 1, wherein the immune response comprises a host innate immune response and/or a host adaptive immune response.

3. The method of claim 1, wherein the immune response comprises the production of at least one antibody capable of binding to the inducer vehicle.

4. The method of claim 1, wherein the immune response comprises the generation of at least one antibody capable of binding to and neutralizing the inducer vehicle.

5. The method of claim 3 or 4, wherein the at least one antibody comprises one or more antibodies that are cross-reactive with the target agent.

6. The method of any one of the preceding claims, wherein the inducer vehicle comprises an inducer viral particle and the target vehicle comprises a target viral particle.

7. The method of claim 6, wherein each of the inducer viral particle and the target viral particle comprises a capsid.

8. The method of claim 7, wherein the immune response comprises the generation of at least one antibody capable of binding to the capsid of the inducer viral particle.

9. The method of claim 8, wherein the at least one antibody is cross-reactive with the capsid of the target viral particle.

10. The method of any one of claims 6 to 9, wherein both the inducer viral particle and the target viral particle are viruses of the same type.

11. The method of claim 10, wherein the type of virus is selected from the group consisting of: adenoviruses, adeno-associated viruses (AAV), lentiviruses, retroviruses, and oncolytic viruses.

12. The method of claim 11, wherein the type of virus is an adeno-associated virus (AAV).

13. The method of any one of claims 6 to 12, wherein the inducer viral particle and the target viral particle are of the same serotype or of different serotypes.

14. The method of any one of claims 6 to 13, wherein each of the inducer viral particle and the target viral particle comprises the same capsid protein.

15. The method according to any one of the preceding claims, wherein the at least one immunosuppressant acts by downregulating an immunostimulatory pathway and/or by upregulating an immunosuppressive pathway.

16. The method of claim 15, wherein one or more of the at least one immunosuppressant reduces expression or activity of one or more immunostimulatory modulators in the immunostimulatory pathway, and optionally wherein each of the one or more immunostimulatory modulators is selected from the group consisting of: CD27, CD70, CD28, CD80 (B7-1), CD86 (B7-2), CD40L (CD 154), CD122, CD137L, OX (CD 134), OX40L (CD 252), GITR, ICOS (CD 278) and ICOSLG (CD 275).

17. The method of claim 16, wherein the one or more immunostimulatory modulators are in at least one of a B7/CD28 or CD40/CD40L costimulatory signaling pathway.

18. The method of claim 17, wherein each of the one or more immunostimulatory modulators is selected from the group consisting of: CD28, CD80 (B7-1), CD86 (B7-2), CD40 and CD40L (CD 154).

19. The method of claim 15, wherein one or more of the at least one immunosuppressant increases expression or activity of one or more immunosuppressant modulators in the immunosuppression pathway, and optionally wherein each of the one or more targets is selected from the group consisting of: a2AR, B7-H3 (CD 276), B7-H4 (VTCN 1), BTLA (CD 272), CTLA-4 (CD 152), IDO1, IDO2, TDO, KIR, LAG3, NOX2, PD-1, PD-L2, TIM-3, VISTA, SIGLEC7 (CD 328), TIGIT, PVR (CD 155) and SIGLEC9 (CD 329).

20. The method of claim 15, wherein the at least one immunosuppressant modulates the expression or activity of at least one of TLR2, TLR9, myD88, IFN-1, IRF-7, NF- κ B, mTOR, CD3, CD4, CD8, CD278, CD19, CD20, CD79, IL-4, IL-2R, IL-5, IL-6R, TNF-a, or LFA-1.

21. The method of claim 15, wherein the at least one immunosuppressant down-regulates the B7-CD28 immunostimulatory pathway, and/or down-regulates CD40 and CD40L immunostimulatory pathways.

22. The method of claim 21, wherein the at least one immunosuppressant inhibits binding or signaling of CTLA-4 and CD80 (B7-1) or CD86 (B7-2); and/or inhibit the binding or signaling of CD40 and CD40L (CD 154).

23. The method of claim 22, wherein the at least one immunosuppressant competes with CD28 for binding to B7, or competes with B7 for binding to CD 28.

24. The method of claim 22, wherein the at least one immunosuppressant competes with CD40 for binding to CD40L, or competes with CD40L for binding to CD 40.

25. The method of any one of the preceding claims, wherein the at least one immunosuppressant comprises at least one immunosuppressive protein.

26. The method according to any one of the preceding claims, wherein the at least one immunosuppressive protein comprises a CTLA-4 derivative and/or a CD40 derivative.

27. The method of claim 26, wherein the at least one immunosuppressive protein comprises:

an extracellular domain of CTLA-4 or a fragment thereof capable of binding to at least one of CD80 (B7-1) or CD86 (B7-2); and/or

The extracellular domain of CD40 or a fragment thereof capable of binding to CD40L (CD 154).

28. The method of claim 26 or claim 27, wherein each immunosuppressive protein of the at least one immunosuppressive protein further comprises a half-life extending peptide moiety.

29. The method of claim 28, wherein the half-life extending peptide moiety comprises Fc.

30. The method of any one of the preceding claims, wherein the at least one immunosuppressant comprises at least one immunosuppressive nucleic acid.

31. The method according to any of the preceding claims, wherein step a) further comprises: administering at least one additional inducer vehicle to the subject, wherein each of the at least one additional inducer vehicle comprises an inducer nucleic acid vector encoding at least one additional immunosuppressant.

32. The method of claim 31, wherein each of the at least one immunosuppressant and the at least one additional immunosuppressant acts on a different immunomodulator.

33. The method according to any of the preceding claims, wherein the step a) further comprises: administering to the subject at least one immunosuppressive agent, each immunosuppressive agent capable of inhibiting or enhancing the activity of a target in an immunostimulatory pathway.

34. The method of claim 33, wherein each of the at least one immunosuppressive agent comprises a compound, a nucleic acid, a polypeptide, or a combination thereof.

35. The method of claim 33 or claim 34, wherein each of the at least one immunosuppressant and the at least one immunosuppressant agent has a different target.

36. The method of any one of the preceding claims, wherein the targeting agent comprises a target nucleic acid vector encoding at least one target gene.

37. The method of claim 36, wherein each of the at least one target gene encodes a therapeutic protein or therapeutic nucleic acid.

38. The method of any one of the preceding claims, wherein the subject has no pre-existing immune response to the inducer vehicle or the target vehicle or both.

39. The method of claim 38, wherein the pre-existing immune response comprises an immune response to a capsid of an inducer viral particle of the inducer vehicle or to a capsid of a target viral particle of the target vehicle or both.

40. The method of claim 38 or claim 39, wherein the pre-existing immune response is characterized by an antibody titer of anti-mediator antibodies of the serum of the subject of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 prior to step a), and wherein the antibody titer is defined as the dilution factor by which the serum of the subject produces a threshold specific binding to the inducer mediator or the target particle.

41. The method of claim 39 or 40, wherein the pre-existing immune response is characterized by an antibody titer of at least 2, 4, 6, 8, 18, or 32 of neutralizing antibodies in the serum of the subject prior to step a), and wherein the antibody titer is defined as the dilution factor by which the serum of the subject produces 50% maximum neutralization of the inducer vehicle or the target vehicle.

42. The method of any one of the preceding claims, wherein the immune tolerance is characterized by an antibody titer of the anti-mediator antibodies of the serum of the subject after step a) of no more than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200, and wherein the antibody titer is defined as the dilution factor by which the serum of the subject produces a threshold specific binding to the inducer mediator or the target mediator.

43. The method according to any one of the preceding claims, wherein the immune tolerance is characterized by an antibody titer of neutralizing antibodies of the serum of the subject after step a) of no more than 2, 4, 6, 8, 18 or 32, and wherein the antibody titer is defined as the dilution factor by which the serum of the subject produces 50% maximum neutralization of the inducer vehicle or the target vehicle.

44. A method of expressing a target gene in a subject by an immunogenic target agent comprising a target vector encoding the target gene, wherein the subject is tolerogenic to the immunogenic target agent, the method comprising:

a) Administering the immunogenic target agent to the subject;

wherein:

prior to step a), the existing immune tolerance has been induced by exposing the subject to an immunogenic inducer vector comprising an inducer nucleic acid vector encoding at least one immunosuppressant, whereby the at least one immunosuppressant has been expressed in the subject to allow suppression of an immune response directed against the inducer vector and having cross-reactivity to a target viral vector.

45. A method of expressing a target gene in a subject by an immunogenic target agent comprising a target vector encoding the target gene, the method comprising:

46. A method of treating a condition in a subject with an immunogenic target agent comprising a target vector encoding a target gene capable of treating the condition, wherein the subject has an existing immune tolerance to the target agent, the method comprising:

a) Administering to the subject an effective amount of the target agent, thereby expressing the target gene in the subject to treat the condition;

wherein:

prior to step a), the existing immune tolerance has been induced by exposing the subject to an immunogenic inducer vehicle comprising an inducer nucleic acid vector encoding at least one immunosuppressant, whereby the at least one immunosuppressant has been expressed in the subject to allow suppression of an immune response directed against the inducer vehicle and cross-reactive with the target vehicle.

47. A method of treating a condition in a subject with an immunogenic target agent comprising a target vector encoding a target gene capable of treating the condition, the method comprising:

a) Administering to the subject an effective amount of an immunogenic inducer vehicle comprising an inducer nucleic acid vector encoding at least one immunosuppressant, thereby allowing expression of the at least one immunosuppressant in the subject, the at least one immunosuppressant inhibiting an immune response directed against the inducer vehicle and being cross-reactive with the target vehicle such that immune tolerance is induced in the subject against the target vehicle; and

b) Administering an effective amount of the target agent to the subject having the induced immune tolerance, thereby expressing the target gene in the subject to treat the condition.

48. The method of any one of claims 44-47, wherein the administering the target agent to the subject comprises repeatedly administering the target agent to the subject.

49. A kit for inducing immune tolerance to an immunogenic target agent to be administered in a subject, the kit comprising:

a first pharmaceutical composition comprising a therapeutically effective amount of an inducer vehicle, wherein the inducer vehicle comprises at least one inducer nucleic acid vector, each inducer nucleic acid vector encoding at least one immunosuppressant and being configured to allow expression of the at least one immunosuppressant in the subject, wherein each immunosuppressant of the at least one immunosuppressant is configured to inhibit an immune response in the subject against the inducer vehicle when expressed in the subject, wherein the immune response is cross-reactive with the target vehicle.

50. The kit of claim 49, wherein the inducer vehicle comprises an inducer viral particle and the target vehicle comprises a target viral particle.

51. The kit of claim 50, wherein each of the inducer viral particle and the target viral particle comprises a capsid.

52. The kit of claim 51, wherein the immune response comprises production of at least one antibody capable of binding to the capsid of the inducer viral particle.

53. The kit of claim 52, wherein the at least one antibody is cross-reactive with the capsid of the target viral particle.

54. The kit of any one of claims 50 to 53, wherein both the inducer viral particle and the target viral particle have the same type of virus.

55. The kit of claim 54, wherein the type of virus is selected from the group consisting of: adenoviruses, adeno-associated viruses (AAV), lentiviruses, retroviruses, and oncolytic viruses.

56. The kit of claim 55, wherein the type of virus is an adeno-associated virus (AAV).

57. The kit of any one of claims 50 to 56, wherein the inducer viral particle and the target viral particle are of the same serotype or of different serotypes.

58. The kit of any one of claims 50 to 57, wherein each of the inducer viral particle and the target viral particle comprises the same capsid protein.

59. The kit of any one of claims 49 to 58, wherein the at least one immunosuppressant acts by downregulating an immunostimulatory pathway and/or by upregulating an immunosuppressive pathway.

60. The kit of claim 59, wherein one or more immunosuppressants of the at least one immunosuppressant reduces expression or activity of one or more targets in the immunostimulatory pathway, and optionally wherein each of the one or more targets is selected from the group consisting of: CD27, CD70, CD28, CD80 (B7-1), CD86 (B7-2), CD40L (CD 154), CD122, CD137L, OX (CD 134), OX40L (CD 252), GITR, ICOS (CD 278) and ICOSLG (CD 275).

61. The kit of claim 60, wherein the one or more targets are in at least one of a B7/CD28 costimulatory signaling pathway or a CD40/CD40L costimulatory signaling pathway.

62. The kit of claim 61, wherein each of the one or more targets is selected from the group consisting of: CD28, CD80 (B7-1), CD86 (B7-2), CD40 or CD40L (CD 154).

63. The kit of claim 59, wherein one or more of the at least one immunosuppressant increases expression or activity of one or more targets in the immunosuppression pathway, and optionally wherein each target of the one or more targets is selected from the group consisting of: a2AR, B7-H3 (CD 276), B7-H4 (VTCN 1), BTLA (CD 272), CTLA-4 (CD 152), IDO1, IDO2, TDO, KIR, LAG3, NOX2, PD-1, PD-L2, TIM-3, VISTA, SIGLEC7 (CD 328), TIGIT, PVR (CD 155) and SIGLEC9 (CD 329).

64. The kit of claim 59, wherein the at least one immunosuppressant modulates the expression or activity of at least one of TLR2, TLR9, myD88, IFN-1, IRF-7, NF- κ B, mTOR, CD3, CD4, CD278, CD19, CD20, CD79, IL-4, IL-2R, IL-5, IL-6R, TNF- α, or LFA-1.

65. The kit of any one of claims 59-64, wherein the at least one immunosuppressant comprises at least one immunosuppressive protein.

66. The kit of claim 65, wherein the at least one immunosuppressive protein comprises:

67. The kit of claim 65 or claim 66, wherein each of the at least one immunosuppressive protein further comprises a half-life extending peptide moiety.

68. The kit of claim 67, wherein the half-life extending peptide moiety comprises Fc.

69. The kit of any one of claims 59 to 64, wherein the at least one immunosuppressant comprises at least one immunosuppressive nucleic acid.

70. The kit of any one of claims 49-69, wherein each of the inducer mediator and the target mediator comprises a viral particle.

71. The kit of claim 70, wherein the viral particles are derived from an adeno-associated virus (AAV), and optionally the viral particles are derived from a packaging cell line, optionally a HEK293 cell line or variant thereof.

72. The kit of any one of claims 49-71, further comprising a first instruction for administering the first pharmaceutical composition to the subject.

73. The kit of any one of claims 49-72, further comprising a first detection kit for determining whether to induce the immune tolerance in the subject after administration of the first pharmaceutical composition to the subject.

74. The kit of claim 73, wherein the immune response comprises production of at least one anti-mediator antibody or at least one neutralizing antibody capable of binding to the inducer mediator and having cross-reactivity with the target mediator, wherein the first detection kit comprises at least one first reagent configured to allow determination of the titer of one or more of the anti-mediator antibodies and/or one or more neutralizing antibodies in the serum of the subject.

75. The kit of claim 73, further comprising a second detection kit, wherein the second detection kit comprises at least one second reagent configured to allow detection of expression of each of the at least one immunosuppressant in the subject.

76. The kit of any one of claims 49-75, further comprising a second pharmaceutical composition, wherein the second pharmaceutical composition comprises a therapeutically effective amount of the target agent.

77. The kit of claim 76, further comprising a second instruction for administering the second pharmaceutical composition to the subject, wherein the second instruction further comprises a first sub-instruction for administering the second pharmaceutical composition to the subject after administration of the first pharmaceutical composition.

78. The kit of claim 77, wherein said second instructions further comprise a second sub-instruction for repeated administration of said second pharmaceutical composition.

79. The kit of any one of claims 76-78, wherein the targeting agent comprises a targeting vector, wherein the targeting vector encodes a target gene and is configured to allow expression of the target gene in the subject.

80. The kit of claim 79, further comprising a third detection kit, wherein the third detection kit comprises at least one third reagent configured to allow detection of expression of the target gene in the subject.

81. A kit for expressing a target gene in a subject, the kit comprising:

a pharmaceutical composition comprising an immunogenic target agent, wherein the target agent comprises a target vector encoding the target gene and configured to allow expression of the target gene in the subject; and

instructions for administering the pharmaceutical composition, the instructions comprising a method for inducing immune tolerance to the target agent prior to administering the pharmaceutical composition to the subject, wherein the method comprises:

a) Administering an immunogenic inducer vehicle to the subject, wherein the inducer vehicle comprises an inducer nucleic acid vector encoding at least one immunosuppressant, thereby allowing expression of the at least one immunosuppressant in the subject, the at least one immunosuppressant inhibiting an immune response in the subject against the inducer vehicle, wherein the immune response is cross-reactive with the target vehicle.

82. The kit of claim 81, further comprising a first detection kit for determining whether to induce the immune tolerance to the target agent in the subject.

83. The kit of claim 82, wherein the immune response comprises production of at least one anti-mediator antibody or at least one neutralizing antibody capable of binding to the inducer mediator and having cross-reactivity with the target mediator, wherein the first detection kit comprises at least one first reagent configured to allow determination of the titer of one or more of the anti-mediator antibodies and/or one or more neutralizing antibodies in the serum of the subject.

84. The kit of any one of claims 80-83, further comprising a second detection kit, wherein the second detection kit comprises at least one second reagent configured to allow detection of expression of each of the at least one immunosuppressant in the subject.

85. The kit of any one of claims 80-84, further comprising a third detection kit, wherein the third detection kit comprises at least one third reagent configured to allow detection of expression of the target gene in the subject.

86. The kit of any one of claims 80-85, wherein the instructions further comprise sub-instructions for repeated administration of the pharmaceutical composition.

87. A kit for expressing a target gene in a subject by an immunogenic target vector encoding the target gene, the kit comprising:

a first pharmaceutical composition for inducing immune tolerance to the target agent in the subject; and

A second pharmaceutical composition comprising the target agent, wherein the target agent comprises a target vector encoding the target gene;

wherein:

the first pharmaceutical composition comprises an inducer vehicle comprising at least one inducer nucleic acid vector, each inducer nucleic acid vector encoding at least one immunosuppressant and configured to allow expression of the at least one immunosuppressant in the subject, wherein each immunosuppressant of the at least one immunosuppressant is configured to inhibit an immune response in the subject against the inducer vehicle when expressed in the subject, wherein the immune response is cross-reactive with the target vehicle.

88. The kit of claim 87, further comprising a first detection kit for determining whether to induce the immune tolerance in the subject following administration of the first pharmaceutical composition to the subject.

89. The kit of claim 88, wherein the immune response comprises production of at least one anti-mediator antibody or at least one neutralizing antibody capable of binding to the inducer mediator and cross-reacting with the target mediator, and the first detection kit comprises At least one first reagentThe at least one first reagent is configured to allow determination of the titer of one or more of the anti-mediator antibodies and/or one or more of the neutralizing antibodies in the serum of the subject.

90. The kit of any one of claims 87 to 89, further comprising a second detection kit, wherein the second detection kit comprises at least one second reagent configured to allow detection of expression of each of the at least one immunosuppressant in the subject.

91. The kit of any one of claims 87 to 90, further comprising a third detection kit, wherein the third detection kit comprises at least one third reagent configured to allow detection of expression of the target gene in the subject.

92. The kit of any one of claims 87 to 91, further comprising:

a first instruction for administering the first pharmaceutical composition; and

a second instruction for administering the second pharmaceutical composition.

93. The kit of claim 92, wherein the second instructions further comprise a first sub-instruction for administering the second pharmaceutical composition to the subject after administration of the first pharmaceutical composition.

94. The kit of claim 92 or claim 93, wherein the second sub-instructions further comprise a second sub-instruction for repeated administration of the second pharmaceutical composition.

95. The kit of any one of claims 49-94, further comprising a third pharmaceutical composition comprising at least one immunosuppressive agent, each immunosuppressive agent capable of inhibiting or enhancing the activity of a target in an immunostimulatory pathway.

96. The kit of claim 95, wherein each of the at least one immunosuppressive agent comprises a compound, a nucleic acid, a polypeptide, or a combination thereof.

97. The kit of claim 95 or claim 96, wherein each of the at least one immunosuppressant and the at least one immunosuppressant agent has a different target.